Topical compositions and methods of using the same

ABSTRACT

The present invention relates generally to compositions and methods of using the same. A first aspect of the present invention comprises a composition comprising a first viscosity increasing agent; at least one polyhydric alcohol; at least one buffering agent; at least one preservative; a second viscosity increasing agent; at least one organic solvent; at least one humectant; at least one active pharmaceutical ingredient; and water, wherein the composition is buffered to a pH of about 3 to about 11.

RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. ProvisionalApplication Ser. No. 61/770,615, filed Feb. 28, 2013, the disclosure ofwhich is hereby incorporated by reference herein in its entirety.

FIELD

The present invention relates generally to compositions and methods ofusing the same.

BACKGROUND

Skin disorders may be topically treated with various pharmaceuticalcompositions.

The present invention addresses previous shortcomings in the art byproviding topical compositions and methods of using the topicalcompositions.

SUMMARY

A first aspect of the present invention comprises a compositioncomprising a first viscosity increasing agent; at least one polyhydricalcohol; at least one buffering agent; at least one preservative; asecond viscosity increasing agent; at least one organic solvent; atleast one humectant; at least one active pharmaceutical ingredient; andwater, wherein the composition is buffered to a pH of about 3 to about11. In some embodiments, the pH of the composition may be about 3 toabout 8. The composition may be cosmetically elegant.

A second aspect of the present invention comprises a compositioncomprising at least one polyhydric alcohol present in an amount of about1% to about 30% by weight of the composition; at least one viscosityincreasing agent present in an amount of about 0.1% to about 5% byweight of the composition; water present in an amount of about 70% toabout 99% by weight of the composition; at least one buffering agentpresent in an amount of about 0.01% to about 2% by weight of thecomposition; and at least one preservative present in an amount of about0.01% to about 1% by weight of the composition; wherein the compositionis buffered to a pH of about 3 to about 8. The composition may becosmetically elegant. [00071 A further aspect of the present inventioncomprises a kit comprising a first composition comprising at least onepolyhydric alcohol present in an amount of about 1% to about 30% byweight of the composition; at least one viscosity increasing agentpresent in an amount of about 0.1% to about 5% by weight of thecomposition; and water present in an amount of about 70% to about 99% byweight of the composition; and a second composition, wherein the secondcomposition is anhydrous. The composition may be cosmetically elegant.

Another aspect of the present invention comprises a method of increasingthe release of nitric oxide from an anhydrous topical gel containing anitric oxide-releasing active pharmaceutical ingredient comprising:contacting the anhydrous topical gel with a first composition of thepresent invention (e.g., a hydrogel of the present invention) having apH of about 4 to about 6 to provide a combined composition. In someembodiments, the combined composition may be applied to the skin of asubject. The combined composition may be cosmetically elegant. Thenitric oxide-releasing active pharmaceutical ingredient may be adiazeniumdiolate modified macromolecule.

A further aspect of the present invention comprises a pharmaceuticalcomposition comprising: an anhydrous topical gel comprising a moisturesensitive active pharmaceutical ingredient; and a first composition ofthe present invention (e.g., a hydrogel of the present invention)comprising means for reducing the pH of the anhydrous topical gel. Themoisture sensitive active pharmaceutical ingredient may be a nitricoxide-releasing active pharmaceutical ingredient and the nitricoxide-releasing active pharmaceutical ingredient may be adiazeniumdiolate modified polysiloxane molecule.

Another aspect of the present invention comprises a method of treatingacne vulgaris comprising topically applying a composition of the presentinvention to the skin of a subject. A therapeutically effective amountof the composition may be applied.

A further aspect of the present invention comprises a method of reducinginflammatory and/or noninflammatory lesions in a subject comprisingtopically applying a composition of the present invention to the skin ofthe subject. A therapeutically effective amount of the composition maybe applied. In some embodiments, the composition may comprise a nitricoxide-releasing active pharmaceutical ingredient in an amount of about0.5% to about 10% by weight of the composition. The composition maystore and/or release nitric oxide in an amount of about 0.05% to about3% by weight of the composition. The method may reduce inflammatoryand/or noninflammatory lesions by about 10% or greater over a definedperiod of time compared to a subject who did not apply a composition ofthe present invention over the same time period. In some embodiments,the method may reduce inflammatory and/or noninflammatory lesions in asubject compared to a subject who did not apply a composition comprisinga nitric oxide-releasing active pharmaceutical ingredient over the sameperiod of time. The subject may see a reduction in inflammatory and/ornoninflammatory lesions within 12 weeks or less, in some embodiments,within 8 weeks or less, and in further embodiments, within 4 weeks orless.

Another aspect of the present invention comprises a method of reducingP. acnes counts in a subject comprising topically applying a compositionof the present invention to the skin of the subject. A therapeuticallyeffective amount of the composition may be applied. In some embodiments,the composition may comprise a nitric oxide-releasing activepharmaceutical ingredient in an amount of about 0.5% to about 10% byweight of the composition. The composition may store and/or releasenitric oxide in an amount of about 0.05% to about 3% by weight ofcomposition. The method may reduce P. acnes counts by about 10% orgreater over a defined period of time compared to a subject who did notapply a composition of the present invention over the same time period.In some embodiments, the method may reduce P. acnes counts in a subjectcompared to a subject who did not apply a composition comprising anitric oxide-releasing active pharmaceutical ingredient over the sameperiod of time. The subject may see a reduction in P. acnes countswithin 12 weeks or less, in some embodiments, within 8 weeks or less,and in further embodiments, within 4 weeks or less.

The foregoing and other aspects of the present invention will now bedescribed in more detail with respect to other embodiments describedherein. It should be appreciated that the invention can be embodied indifferent forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph of the effects of Nitricil™ NVN1 Topical Gelstrength, hydrogel pH, and hydrogel to Nitricil™ NVN1 Topical Gel ratioon the admixture pH.

FIG. 2 shows a graph of the in vitro pH determination for Nitricil™ NVN1topical gels when admixed with an unbuffered or buffered (0.1% citricacid) hydrogel (pH 4).

FIG. 3 shows a graph of the skin surface pH determination for admixturesof an unbuffered or buffered pH 4 hydrogel and an 8% w/w Nitricil™ NVN1Topical Gel.

FIG. 4 shows a graph of the effects of Nitricil™ NVN1 Topical Gelstrength and hydrogel ratio on C_(max).

FIG. 5 shows a graph of the effects of Nitricil™ NVN1 Topical Gelstrength and hydrogel ratio on cumulative nitric oxide (NO) release.

FIG. 6 shows a graph of the effects of unbuffered hydrogel pH and ratioon admixture pH with respect to Nitricil™ NVN1 Topical Gel strength.

FIG. 7 shows the in vitro nitric oxide release profile for a 2%Nitricil™ NVN1 Gel formulation over time.

FIG. 8 shows the in vitro nitric oxide release profiles for 2%, 6%, and12% Nitricil™ NVN1 Gel formulations upon mixing with the hydrogel at pH4 over time.

FIG. 9 shows a graph of the change in P. acnes counts (log/cm²) overtime based on an analysis of co-variance (ANCOVA).

FIG. 10 shows a graph of the mean percentage reduction over time fornoninflammatory lesions in the per-protocol population.

FIG. 11 shows a graph of the mean percentage reduction over time forinflammatory lesions in the per-protocol population.

FIG. 12 shows a graph of the cumulative nitric oxide release for a 2%Nitricil™ NVN1 Gel without a hydrogel and a 1% Nitricil™ NVN1 Gelcontaining a hydrogel (the gel to hydrogel ratio was 1:1).

DETAILED DESCRIPTION

The present invention will now be described more fully hereinafter. Thisinvention may, however, be embodied in different forms and should not beconstrued as limited to the embodiments set forth herein. Rather, theseembodiments are provided so that this disclosure will be thorough andcomplete, and will fully convey the scope of the invention to thoseskilled in the art.

The terminology used in the description of the invention herein is forthe purpose of describing particular embodiments only and is notintended to be limiting of the invention. As used in the description ofthe invention and the appended claims, the singular forms “a”, “an” and“the” are intended to include the plural forms as well, unless thecontext clearly indicates otherwise.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the present applicationand relevant art and should not be interpreted in an idealized or overlyformal sense unless expressly so defined herein. The terminology used inthe description of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety. In case of a conflict in terminology, the presentspecification is controlling.

Also as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

Unless the context indicates otherwise, it is specifically intended thatthe various features of the invention described herein can be used inany combination. Moreover, the present invention also contemplates thatin some embodiments of the invention, any feature or combination offeatures set forth herein can be excluded or omitted. To illustrate, ifthe specification states that a complex comprises components A, B and C,it is specifically intended that any of A, B or C, or a combinationthereof, can be omitted and disclaimed.

As used herein, the transitional phrase “consisting essentially of” (andgrammatical variants) is to be interpreted as encompassing the recitedmaterials or steps “and those that do not materially affect the basicand novel characteristic(s)” of the claimed invention. See, In re Herz,537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (COPA 1976) (emphasis in theoriginal); see also MPEP §2111.03. Thus, the term “consistingessentially of” as used herein should not be interpreted as equivalentto “comprising.”

The term “about,” as used herein when referring to a measurable value,such as an amount or concentration and the like, is meant to refer tovariations of up to ±20% of the specified value, such as, but notlimited to, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified value,as well as the specified value. For example, “about X” where X is themeasurable value, is meant to include X as well as variations of ±20%,±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of X. A range provided herein for ameasureable value may include any other range and/or individual valuetherein.

According to some embodiments of the present invention, provided hereinare topical compositions. A composition of the present invention maycomprise at least two parts. In some embodiments, a composition of thepresent invention comprises a first part comprising a first compositionand a second part comprising a second composition. The second part of acomposition of the present invention may comprise a nitricoxide-releasing active pharmaceutical ingredient (NO-releasing API). Insome embodiments, a composition of the present invention may comprise afirst part comprising a first composition that may be in the form of ahydrogel. “Hydrogel,” as used herein, refers to a hydrophilic gelcomprising a gel matrix and water. In some embodiments, a firstcomposition of the present invention comprises at least one polyhydricalcohol, at least one viscosity increasing agent, and water.

In particular embodiments, a composition of the present inventioncomprises, consists essentially of, or consists of a first compositionof the present invention and a second composition, wherein the firstcomposition and the second composition are compatible with each othersuch that they may be mixed and/or combined together to provide thecomposition. A composition of the present invention may be referred toas a combined composition in some embodiments. In further aspects, thecombined composition may be suitable for topical application to the skinof a subject.

Exemplary polyhydric alcohols that may be present in a first compositionof the present invention include, but are not limited to, glycerol,propylene glycol, polyethylene glycol, polypropylene glycol, triethyleneglycol, neopental glycols, butylene glycol, polyethylene glycol,sorbitol, arabitol, erythritol, HSH, isomalt, lactitol maltitol,mannitol, xylitol, threitol, ribitol, galactitol, fucitol, iditol,inositol, volemitol, and any combination thereof. In some embodiments, afirst composition of the present invention comprises glycerol, such as,but not limited to, anhydrous glycerol.

A polyhydric alcohol may be present in a first composition of thepresent invention in an amount of about 1% to about 30% by weight of thefirst composition or any range and/or individual value therein, such as,but not limited to, about 1% to about 20%, about 1% to about 10%, orabout 5% to about 15% by weight of the first composition. In certainembodiments, a polyhydric alcohol may be present in a first compositionof the present invention in an amount of about 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%,22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% by weight of the firstcomposition or any range and/or individual value therein.

Exemplary viscosity increasing agents that may be present in a firstcomposition of the present invention include, but are not limited to, acarboxypolymethylene; a polyacrylic polymer such as polyacrylic acid, apolyacrylate polymer, a cross-linked polyacrylate polymer, across-linked polyacrylic acid, and mixtures thereof; a cellulose ethersuch as hydroxyalkyl cellulose polymers such as hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose, hyrdoxyethyl cellulose,methyl cellulose, carboxymethyl cellulose, and mixtures thereof; amethacrylate; a polyvinylpyrollidone; cross-linked polyvinylpyrrolidone; polyvinylpyrrolidone-vinyl acetate copolymer;polyvinylalcohol; polyethylene oxide; polyethylene glycol;polyvinylalkyl ether-maleic acid copolymer; a carboxy vinyl polymer; apolysaccharide; a gum such as sodium alginate, carrageenan, xantham gum,gum acacia, arabic gum, guar gum, pullulan, agar, chitin, chitosan,pectin, karaya gum, zein, hordein, gliadin, locust bean gum,tragacantha, and mixtures thereof; a protein such as collagen, wheyprotein isolate, casein, milk protein, soy protein, gelatin, andmixtures thereof; a starch such as maltodextrin, amylose, high amylasestarch, corn starch, potato starch, rice starch, tapioca starch, peastarch, sweet potato starch, barley starch, wheat starch, waxy cornstarch, modified starch (e.g. hydroxypropylated high amylose starch),dextrin, levan, elsinan, gluten, and mixtures thereof; bentonite;calcium stearate; ceratonia; colloidal silicon dioxide; dextrin;hypromellose; polycarbophil; kaolin; saponite; sorbitan esters; sucrose;sesame oil; tragacanth; potassium alginate; povidone; sodium starchglycolate; phospholipids; and any combination thereof.

In some embodiments, a first composition of the present inventioncomprises a carboxypolymethylene, such as, but not limited to, thosecommercially available from Lubrizol Corporation of Wickliffe, Ohiounder the trade name Carbopol®. Exemplary Carbopol® polymers that may bepresent in a first composition of the present invention include, but arenot limited to, Carbopol® 974P NF polymer, such as Type A, Type B and/orType C Homopolymers; Carbopol® Ultrez 10, 20, 21 NF polymer; Carbopol®971 P NF polymer; Carbopol® 980 Homopolymer Type C polymer, Carbopol®980 NF polymer, Carpobol® 980P polymer, Carbopol® ETD 2020 NF polymer,Carbopol® 71 G NF polymer, Carbopol® 981P NF polymer, Carbopol® 970P NFpolymer, Carbopol® 981P NF polymer, Carbopol® 5984P NF polymer,Carbopol® 934P NF polymer, Carbopol® 940P NF polymer, Carbopol® 941P NFpolymer, Carbopol® 13242 NF polymer, Carbopol® AA-1 USP NF polymer,Carbopol® TR1 NF polymer, Carbopol® TR2 NF polymer, Lubrizol Aqua CCpolymer and SF-2 polymer, and any combination thereof.

In some embodiments, a viscosity increasing agent present in a firstcomposition of the present invention may be a polymer comprising acidicgroups, such as, but not limited to, carboxylic acid groups. The acidicgroups of the polymer may be partially neutralized in a firstcomposition of the present invention. In certain embodiments, aviscosity increasing agent present in a first composition of the presentinvention may be a carboxypolymethylene. In some embodiments, acarboxypolymethylene present in a first composition of the presentinvention may be partially neutralized. A first composition of thepresent invention may comprise a carboxypolymethylene and have a pH ofabout 3 to about 7, about 3.5 to about 6.5, about 3.5 to about 6, orabout 4 to about 6. In certain embodiments, a first composition of thepresent invention may comprise a carboxypolymethylene and have a pH ofabout 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7.

A viscosity increasing agent may be present in a first composition ofthe present invention. In some embodiments, a composition of the presentinvention may comprise at least two viscosity increasing agents that maybe the same or different. In some embodiments, a first viscosityincreasing agent may be present in a first composition of the presentinvention in an amount of about 0.01% to about 5% by weight of the firstcomposition or any range and/or individual value therein, such as, butnot limited to, about 0.05% to about 3% or about 0.1% to about 1.5% byweight of the first composition. In certain embodiments, a firstviscosity increasing agent is present in a first composition of thepresent invention in an amount of about 0.01%, 0.02%, 0.03%, 0.04%,0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%,0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5% byweight of the first composition or any range and/or individual valuetherein.

Water may be present in a first composition of the present invention inan amount of about 70% to about 99% by weight of the first compositionor any range and/or individual value therein, such as, but not limitedto, about 75% to about 95% or about 80% to about 90% by weight of thefirst composition. In certain embodiments, water is present in a firstcomposition of the present invention in an amount of about 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%by weight of the first composition or any range and/or individual valuetherein.

In some embodiments, a first composition of the present inventioncomprises, consists essentially of, or consists of at least onepolyhydric alcohol present in an amount of about 1% to about 30% byweight of the first composition, at least one viscosity increasing agentpresent in an amount of about 0.1% to about 5% by weight of the firstcomposition, and water present in an amount of about 70% to about 99% byweight of the first composition. In certain embodiments, the viscosityincreasing agent may be a carboxypolymethylene. The first compositionmay be a hydrogel.

A first composition of the present invention may comprise apreservative. A preservative may be present in a first composition ofthe present invention in an amount of about 0.01% to about 1% by weightof the first composition or any range and/or individual value therein,such as, but not limited to, about 0.01% to about 0.1%, about 0.05% toabout 1%, or about 0.1% to about 1% by weight of the first composition.In certain embodiments, a preservative is present in a first compositionof the present invention in an amount of about 0.01%, 0.02%, 0.03%,0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%,0.6%, 0.7%, 0.8%, 0.9%, or 1% by weight of the first composition or anyrange and/or individual value therein. Exemplary preservatives that maybe present in a first composition of the present invention include, butare not limited to, sorbic acid, benzoic acid, methyl-paraben,propyl-paraben, methyl chloroisothiazolinone, metholisothiazolinone,diazolidinyl urea, chlorobutanol, triclosan, benzethonium chloride,p-hydroxybenzoate, chlorhexidine, digluconate, hexadecyltrimethylammonium bromide, alcohols, benzalkonium chloride, boric acid, bronopol,butylparaben, butylene calcium acetate, calcium chloride, calciumlactate, carbon dioxide, cationic, and bentonite, cetrimide,cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol,chloroxylenol, citric acid monohydrate,cresol, dimethyl ether,ethylparaben, glycerin, hexetidine, imidurea, isopropyl alcohol, lacticacid, monothioglycerol, pentetic acid, phenol, phenoxyethanol,phenylethyl alcohol, phenylmercuric acetate, phenylmercuric borate,phenylmercuric nitrate, potassium benzoate, potassium metabisulfite,potassium sorbate, propionic acid, propyl gallate, propylene glycol,sodium acetate, sodium benzoate, sodium borate, sodium lactate, sodiumsulfite, sodium propionate, sodium metabisulfite, xylitol, sulphurdioxide, carbon dioxide,and any combination thereof.

A first composition of the present invention may comprise a neutralizingagent. A neutralizing agent may be present in a first composition of thepresent invention in an amount sufficient to provide a desired pH, suchas, but not limited to, a pH of about 3 to about 11, or any range and/orindividual value therein, such as, but not limited to, about 3 to about8, about 4 to about 7, or about 6 to about 7, or about 6 to 11.

In some embodiments, a neutralizing agent may be present in a firstcomposition in an amount sufficient to provide the first compositionwith a pH between about 3 to about 8.

In certain embodiments, a neutralizing agent may be present in a firstcomposition of the present invention in an amount sufficient to providea composition of the present invention with a desired pH uponcombination of the first composition and a second part (e.g., a secondcomposition) and/or upon administration of the first composition and/orthe composition comprising the first composition and the second part tothe skin of a subject. A neutralizing agent may be present in a firstcomposition of the present invention in an amount sufficient to providea composition of the present invention (e.g., a composition comprisingthe first composition and a second composition) with a desired pH, suchas, but not limited to, a pH of about 3 to about 11, or any range and/orindividual value therein.

The pH of a composition of the present invention may be determined oncea steady state pH is achieved after contact, combination, and/or mixingof the first part and second part of the composition of the presentinvention. Alternatively or in addition, the pH of a composition of thepresent invention may be determined after a defined period of time, suchas, but not limited to, after about 5, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60 minutes or more. The pH of a composition of the presentinvention may be measured in vitro after contact, combination, and/ormixing of the first part and second part of a composition of the presentinvention. Alternatively or in addition, the pH of a composition of thepresent invention may be measured after administration to a subject,such as, for example, a skin surface pH may be measured afteradministration of a composition of the present invention to the skin ofa subject.

In some embodiments, a neutralizing agent adjusts the pH of the firstcomposition and/or composition of the present invention. In certainembodiments of the present invention, a neutralizing agent is present ina first composition of the present invention in an amount sufficient forthe first composition to have a pH of about 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, or 8 or any range and/or individual value therein. In someembodiments of the present invention, a neutralizing agent is present ina composition of the present invention in an amount sufficient for thecomposition to have a pH of about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7,7.5, 8, 8.5, 9, 9.5, 10, 10.5, or 11, or any range and/or individualvalue therein.

Exemplary neutralizing agents that may be present in a first compositionof the present invention include, but are not limited to, bases such assodium hydroxide, potassium hydroxide, and mixtures thereof; acids suchas hydrochloric acid, citric acid, lactic acid, glycolic acid, aceticacid, and mixtures thereof; sodium carbonate; trolamine; tromethamine;aminomethyl propanol; triisopropanolamine; aminomethyl propanol;tetrahydroxypropyl ethylenediamine; tetrasodium EDTA; suttocide A; andany combination thereof.

A neutralizing agent may be present in a first composition of thepresent invention in an amount of about 0.01% to about 1% by weight ofthe first composition or any range and/or individual value therein, suchas, but not limited to, about 0.01% to about 0.1%, about 0.05% to about1%, or about 0.1% to about 1% by weight of the first composition. Incertain embodiments, a neutralizing agent may be present in a firstcomposition of the present invention in an amount of about 0.01%, 0.02%,0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%,0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% by weight of the first compositionor any range and/or individual value therein.

A first composition of the present invention may be unbuffered orbuffered. In some embodiments, a first composition of the presentinvention may be unbuffered. In other embodiments, a first compositionof the present invention may be buffered. Exemplary buffers that may bepresent in first composition of the present invention include, but arenot limited to, acetic acid/acetate buffers; hydrochloric acid/citratebuffers; vitro-phosphate buffers; phosphate buffers; citric acid/citratebuffers; lactic acid buffers; tartaric acid buffers; malic acid buffers;glycine/HCI buffers; saline buffers such as phosphate buffered saline(PBS), Tris-buffered saline (TBS), Tris-HCl, NaCl, Tween buffered saline(TNT), phosphate buffered saline, Triton X-100 (PBT) and mixturesthereof; cacodylate buffers; barbital buffers; tris buffers; and anycombination thereof.

In certain embodiments, a first composition of the present invention maycomprise a buffering agent. Exemplary buffering agents include, but arenot limited to, citric acid, acetic acid, lactic acid, boric acid,succinic acid, malic acid, and any combination thereof. A bufferingagent may be present in a first composition of the present invention inan amount of about 0.01% to about 2% by weight of the first compositionor any range and/or individual value therein, such as, but not limitedto, about 0.01% to about 0.1%, about 0.05% to about 1%, about 0.1% toabout 0.5%, or about 0.1% to about 2% by weight of the firstcomposition. In certain embodiments, a buffering agent is present in afirst composition of the present invention in an amount of about 0.01%,0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%,0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%,1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2% by weight of the first compositionor any range and/or individual value therein.

In some embodiments, a buffer and/or buffering agent is present in afirst composition of the present invention in an amount sufficient forthe first composition to have a pH of about 3 to about 8 or any rangeand/or individual value therein, such as, but not limited to, about 3 toabout 6, about 3 to about 5, about 4 to about 7, about 5 to about 7, orabout 6 to about 7. In certain embodiments of the present invention, abuffer and/or buffering agent may be present in a first composition ofthe present invention in an amount sufficient for the first compositionto have a pH of about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8, orany range and/or individual value therein.

In some embodiments, a buffer and/or buffering agent may be present in afirst composition of the present invention in an amount sufficient toprovide a desired pH for a composition of the present inventioncomprising the first composition and a second part (e.g., a secondcomposition). For example, a composition of the present invention maycomprise a second composition and a first composition comprising abuffer and/or buffering agent, wherein the buffer and/or buffering agentis present in an amount sufficient to provide the composition with a pHof about 3 to about 11, such as, but not limited to, about 3 to about 8,about 7 to about 11, about 8 to about 10, about 3 to about 5, about 4 toabout 7, about 5 to about 7, or about 6 to about 7. In certainembodiments of the present invention, a buffer and/or buffering agentmay be present in a first composition of the present invention in anamount sufficient for the composition to have a pH of about 3, 3.5, 4,4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, or 11, or anyrange and/or individual value therein. In some embodiments, the bufferand/or buffering agent may be present in a first composition of thepresent invention in an amount sufficient to provide a desired pH uponadministration of a composition of the present invention comprising thefirst composition and a second part to the skin of a subject.

In some embodiments, a buffer, buffering agent, and/or neutralizingagent may be present in a first composition of the present invention inan amount sufficient to provide a composition of the present inventionand/or a first composition of the present invention with a desired pH.

In certain embodiments, a first composition of the present inventioncomprises at least one polyhydric alcohol present in an amount of about1% to about 30% by weight of the first composition, at least oneviscosity increasing agent present in an amount of about 0.1% to about5% by weight of the first composition, water present in an amount ofabout 70% to about 99% by weight of the first composition, and at leastone preservative in an amount of about 0.01% to about 1% by weight ofthe first composition. The first composition may be buffered to have apH in a range of about 3 to about 8, about 3 to about 6, or about 6 toabout 8. The first composition may be a hydrogel.

In some embodiments, a first composition of the present invention maycomprise, consist essentially of, or consist of a polyhydric alcohol inan amount of about 1% to about 30% by weight of the first composition, aviscosity increasing agent in an amount of about 0.01% to about 5% byweight of the first composition, water in an amount of about 70% toabout 99% by weight of the first composition, optionally a bufferingagent in an amount of about 0.001% to about 2% by weight of the firstcomposition, optionally a preservative in an amount of about 0.001% toabout 1% by weight of the first composition, and optionally aneutralizing agent in an amount of about 0.001% to about 1% by weight ofthe first composition. The first composition may have a pH in a range ofabout 3 to about 5 or about 5 to about 7. In certain embodiments, theviscosity increasing agent present in the first composition may be acarboxypolymethylene. In some embodiments, the first composition may becosmetically elegant. The first composition may be a hydrogel.

As those skilled in the art will recognize in light of the presentdisclosure, the properties of a first composition of the presentinvention may confer and/or provide the same and/or similar propertiesto a composition of the present invention. For example, in someembodiments, a first composition of the present invention may comprise apreservative that is present in an amount sufficient to provideantimicrobial activity to the first composition and/or a composition ofthe present invention. Thus, in some embodiments, a first compositionand/or a composition of the present invention may be antimicrobial.

A composition and/or first composition of the present invention may havea viscosity in a range of about 5,000 cP to about 25,000 cP or any rangeand/or individual value therein, such as, but not limited to, about5,000 cP to about 20,000 cP or about 7,000 cP to about 15,000 cP. Incertain embodiments, a composition and/or first composition of thepresent invention may have a viscosity of about 5,000, 5,500, 6,000,6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500, 10,000, 10,500, 11,000,11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500,16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000,20,500, 21,000, 21,500, 22,000, 22,500, 23,000, 23,500, 24,000, 24,500,or 25,000 cP or any range and/or individual value therein.

In some embodiments, a composition of the present invention may comprisea first composition of the present invention that has a viscosity thatallows for mixing and/or combination with a second part of thecomposition. For example, a first composition of the present inventionmay have a viscosity suitable and/or sufficient for mixing and/orcombination with a second part of a composition of the present inventionin a person's hand and/or on a subject's skin. A first composition withtoo low of a viscosity may run off the skin of a subject prior to mixingand/or combination. A first composition with too high a viscosity may bedifficult to mix with the second part of a composition of the presentinvention and/or difficult to spread and/or apply the combinedcomposition on the skin of a subject.

A composition and/or first composition of the present invention maycomprise an active pharmaceutical ingredient (API). Any suitable API orcombinations of APIs may be included in a composition and/or firstcomposition of the present invention. Examples of APIs include, but arenot limited to, antimicrobial agents, anti-acne agents,anti-inflammatory agents, analgesic agents, anesthetic agents,antihistamine agents, antiseptic agents, immunosuppressants,antihemorrhagic agents, vasodilators, wound healing agents, anti-biofilmagents, and any combination thereof. Exemplary APIs include, but are notlimited to, those described in International Application Publication No.WO 2013/006608, which is incorporated herein by reference in itsentirety.

In some embodiments, a first composition of the present invention maynot comprise an API. In certain embodiments, a first composition of thepresent invention does not contain a nitric oxide (NO) releasing API. Insome embodiments, a first composition of the present invention maycomprise at least one API, but the first composition may not comprise anNO-releasing API.

In certain embodiments, a first composition of the present invention maycomprise an API. The API in the first composition may be a moisturesensitive API. In some embodiments, a first composition of the presentinvention comprises an API (e.g., a moisture sensitive API) and thesecond part and/or composition of a composition of the present inventioncomprises a second API, wherein the API in the first composition and thesecond API in the second part and/or composition may not be chemicallycompatible and/or stable in the composition of the present invention.For example, the API in the first composition and the second API in thesecond part and/or composition may not be chemically compatible and/orstable when stored together in a composition of the present invention.

A first composition of the present invention may be suitable for useand/or combination with one or more, such as, but not limited to, 2, 3,4, or more, compositions that may be the same and/or different. In someembodiments, a first composition of the present invention may besuitable for use and/or combination with one or more pharmaceuticalcompositions. A first composition of the present invention may be usedas a drug delivery system and/or a drug release system. For example, afirst composition of the present invention may be configured to modulatethe release of an API in a second composition upon contact of the firstcomposition of the present invention (e.g., a hydrogel of the presentinvention) and a second composition. Alternatively or in addition, afirst composition of the present invention may be configured to modulatethe pH of a second composition upon contact of the first composition ofthe present invention (e.g., a hydrogel of the present invention) andthe second composition. In some embodiments, a first composition of thepresent invention may be configured to modulate the pH of a secondcomposition comprising a nitric oxide (NO) releasing API and/or therelease of nitric oxide from an NO releasing API in a secondcomposition.

“Modulate,” “modulating,” “modulation,” and grammatical variationsthereof as used herein refer to an increase or reduction in the pH of asecond composition and/or the release of an API in a second compositioncompared to the pH of the second composition and/or the release of theAPI in the second composition in the absence of a first composition ofthe present invention. As used herein, the terms “increase,”“increases,” “increased,” “increasing” and similar terms indicate anelevation in the pH and/or release of at least about 5%, 10%, 25%, 50%,75%, 100%, 150%, 200%, 300%, 400%, 500% or more compared to the pHand/or release in the absence of a first composition of the presentinvention. As used herein, the terms “reduce,” “reduces,” “reduced,”“reduction” and similar tetins refer to a decrease in the pH and/orrelease of at least about 5%, 10%, 25%, 35%, 50%, 75%, 80%, 85%, 90%,95%, 97% or more compared to the pH and/or release in the absence of afirst composition of the present invention.

“Contact,” as used herein in reference to a first composition of thepresent invention (e.g., a hydrogel of the present invention) and asecond part and/or composition, refers to direct and/or indirectexposure of at least one component in the first composition to thesecond part and/or composition. Contact of the first composition andsecond part and/or composition may be accomplished by any means, suchas, but not limited to, by mixing, stirring, blending, dispersing,milling, homogenizing, combining, applying to same area or region, andthe like, and in some embodiments may optionally form a combinedcomposition of the present invention. For example, a first compositionmay come into direct contact with a second composition, such as, but notlimited to, by mixing and/or combining the first composition and secondcomposition to form a combined composition of the present inventionprior to, during, and/or after topical application to a subject. Directcontact of a first composition and second composition may occur byapplying one or more layers of the second composition onto a subject andthen applying one or more layers of the first composition onto a subjector vice versa to form a combined composition of the present invention.Indirect contact may occur by applying a second composition onto asubject and then applying a first composition onto a subject through asubstrate, such as, but not limited to, a cloth, bandage, gauze, and thelike, or vice versa to optionally form a combined composition of thepresent invention.

According to some embodiments of the present invention, upon contact ofa first composition of the present invention and a second composition, afirst composition of the present invention may be configured to modulatethe release of an API present in the second composition, such as, butnot limited to, an NO releasing API. In some embodiments, water and/or aproton(s) present in a first composition of the present invention maycontact a second composition to modulate the release of an API presentin the second composition, such as, but not limited to, an NO releasingAPI. Alternatively or in addition, in some embodiments, contact of afirst composition of the present invention with a second composition maymodulate the pH of the second composition, thereby modulating therelease of an API present in the second composition, such as, but notlimited to, an NO releasing API. In some embodiments, a firstcomposition of the present invention is configured to supply waterand/or a proton(s) to a second composition and/or configured to modulatethe pH of a second composition.

It was surprisingly discovered by the inventors of the present inventionthat embodiments of the present invention could significantly increasethe amount of nitric oxide released from a composition comprising anNO-releasing API and/or provide a continuous release of nitric oxidefrom a composition comprising an NO-releasing API. For example, this canbe seen in FIG. 12, which compares a composition without a firstcomposition of the present invention (i.e., the 2% Nitricil™ NVN1 Gel)and a composition of the present invention comprising a firstcomposition of the present invention (i.e., the 1% Nitricil™ NVN1 Gel).The 2% Nitricil™ NVN1 Gel and the part of 1% Nitricil™ NVN1 Gel with theNO-releasing API contained the same amount of the NO-releasing API. FIG.12 demonstrates that the I% Nitricil™ NVN1 Gel had a significantlyhigher amount of nitric oxide released and also provided for acontinuous release of nitric oxide over a period of time compared to the2% Nitricil™ NVN1 Gel.

While not wishing to be bound to any particular theory, it is believedthat a composition of the present invention comprising a firstcomposition of the present invention and a NO-releasing API may providea proton donating system that may provide for a high release of nitricoxide from the composition and/or a continuous release of nitric oxidefrom the composition. The proton donating system, while not wishing tobe bound to any particular theory, may be an acid that may be formed bya composition of the present invention (e.g., a composition comprising afirst composition of the present invention and a second composition asdescribed herein) and/or a first composition of the present invention(e.g., a hydrogel of the present invention). A composition of thepresent invention may provide and/or allow for a proton to be in closeproximity to an NO-donor in an NO-releasing API to thereby allow for therelease of nitric oxide. A composition of the present invention mayprovide and/or allow for a proton to be in proximity of an NO-donor foran extended period of time to provide a continuous release of NO forabout 1 or more hours, such as, but not limited to, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, or more hours.

The proton donating system may be provided by a first composition of thepresent invention. For example, a proton donating system may be providedby a first composition of the present invention comprising an acidicpolymer, such as, but not limited to, carboxypolymethylene, wherein theacidic polymer is partially neutralized and the first composition has apH of about 3 to about 8 or any range therein, such as, but not limitedto, about 3 to about 6, about 3 to about 6, or about 5 to about 7. Whilenot wishing to be bound to any particular theory, the partiallyneutralized acidic polymer may provide the first composition with apolymeric gel matrix having a coiled structure. The coiled structure ofthe polymeric gel matrix may provide and/or donate protons when theprotons are in proximity to an NO donor in a composition of the presentinvention and the coiled structure may protect some protons (e.g.,interior protons) from reacting with an NO donor in the composition. Asthe polymeric gel matrix uncoils (e.g., as the pH of the compositionbecomes more basic and/or the polymeric gel matrix becomes neutralized),additional protons (e.g., the protected protons) may be in proximity toa NO donor, and this may provide for the release of nitric oxide fromthe composition over a longer period of time and/or for a higher amountof nitric oxide released from the composition.

In particular embodiments, a first composition of the present inventionmodulates the pH of a second composition such that when the first andsecond compositions are contacted and/or applied to the skin of asubject, the pH of the second composition and/or combined composition(i.e., a composition of the present invention) is less that about 11, insome embodiments, less than about 10, in certain embodiments, less thanabout 8.5, in further embodiments, less than about 7, and in stillfurther embodiments, between about 6 and about 8.

In some embodiments, the pH of a combined composition of the presentinvention changes upon application of the combined composition to theskin of a subject. In particular embodiments, the pH of a combinedcomposition of the present invention is decreased by the bufferingcapacity of the skin upon application of the combined composition to theskin of a subject. In some embodiments, the pH of a combined compositionof the present invention after application of the combined compositionto the skin of a subject is less than the pH of the second compositionapplied to the skin without the first composition. In embodiments wherethe release kinetics of the API in a second composition varies with pH,the buffering capacity of the skin may be utilized to modulate releasewhile improving stability of the combined composition after combinationand before application. Thus, for example, the pH of a secondcomposition that includes a nitric oxide-releasing macromolecule may begreater than 10 before mixing, 9 after mixing and 8 after application tothe skin. With each decrease in pH, the release of nitric oxide from themacromolecule may be increased. Accordingly, taking advantage of thechanging pH and buffering capacity of the skin may allow for increasedworking time (e.g., mixing and application time) for a combinedcomposition of the present invention.

In some embodiments, a composition of the present invention may comprisea second composition and the second composition may be an anhydrouscomposition. “Anhydrous,” as used herein, means that there is no directaddition of water to the second composition when it is being prepared.However, those skilled in the art will recognize that water may bephysically and/or chemically absorbed by the second composition and/orby one or more ingredients in the second composition at any time duringthe preparation, storage, and/or use of the second composition (i.e.,indirect addition of water to the second composition). In someembodiments, the term “anhydrous” means that the second composition hasa water content of less than 5% by weight of the second composition orany range and/or individual value therein. A second composition may havea water content of less than 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, or 0.5%,or any range therein, by weight of the second composition. Water contentmay be measured by methods known to those of skill in the art, such as,but not limited to, Karl Fischer titration. In certain embodiments, uponcontact with a second composition, a composition of the presentinvention adds water to the second composition and/or the secondcomposition absorbs water from a composition of the present invention.

Exemplary second compositions that may be used and/or placed in contactwith a first composition of the present invention include, but are notlimited to, those described in International Application Publication No.WO 2013/006608, which is incorporated herein by reference in itsentirety. An exemplary second composition that may be used and/or placedin contact with a first composition of the present invention to form acomposition of the present invention (e.g., a combined composition ofthe present invention) may comprise an anhydrous composition comprisingat least one viscosity increasing agent present in the secondcomposition in an amount of about 0.5% to about 30% by weight of thesecond composition, at least one organic solvent present in the secondcomposition in an amount of about 50% to about 90 by weight of thesecond composition, and at least one humectant present in the secondcomposition in an amount of about 2% to about 20% by weight of thesecond composition. The second composition may further comprise at leastone water repelling agent, also referred to as a water repellant. Incertain embodiments, the second composition may comprise a compositionas set forth in Table 11.

Exemplary viscosity increasing agents for the second compositioninclude, but are not limited to, co-polymers of carboxymethylcelluloseand acrylic acid, N-vinylpyrrolidone, polyalkylene glycols (e.g.,poly(ethylene glycol)), polyalkylene oxides (e.g., polyethylene oxide),polyvinyl alcohols, polyvinylpyrrolidone, polysiloxanes, poly(vinylacetates), cellulose, derivatized celluloses, alginates, copolymersthereof and blends thereof. A specific example of a viscosity agent forthe second composition is a hydroxypropylcellulose, such as Klucel®hydroxypropylcellulose (e.g., Klucel® MF Pharm grade). A viscosityincreasing agent may be present in the second composition in an amountof about 0.1% to about 30% by weight of the second composition or anyrange and/or individual value therein, such as, but not limited to,about 0.5% to about 20%, about 1% to about 10%, or about 1% to about 5%by weight of the second composition. In certain embodiments, a viscosityincreasing agent may be present in the second composition in an amountof about 0.1%, 0.25%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, or 30% by weight of the second compositionor any range and/or individual value therein.

Exemplary organic solvents for the second composition include, but arenot limited to, acetone, methyl alcohol, ethanol, isopropanol, butylalcohol, ethyl acetate, dimethyl isosorbide, propylene glycol, glycerol,ethylene glycol, polyethylene glycol, diethylene glycol monoethyl etheror mixtures thereof. In some embodiments of the present invention, theorganic solvent in the second composition may be ethanol and/orisopropyl alcohol. An organic solvent may be present in the secondcomposition in an amount of about 50% to about 90% by weight of thesecond composition or any range and/or individual value therein, suchas, but not limited to, about 60% to about 90%, about 70% to about 90%,or about 75% to about 85% by weight of the second composition. Incertain embodiments, an organic solvent may be present in the secondcomposition in an amount of about 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, or 90% by weight of the second composition orany range and/or individual value therein.

Exemplary humectants for the second composition include, but are notlimited to, glycols, such as diethylene glycol monoethyl ether;glycerols; sugar polyols, such as sorbitol, xylitol and maltitol;polyols such as polydextroses; quillaia, urea, and blends thereof. Insome embodiments, the humectant in the second composition may comprisean alkylene glycol, such as, for example, hexylene glycol. A humectantmay be present in the second composition in an amount of about 2% toabout 20% by weight of the second composition or any range and/orindividual value therein, such as, but not limited to, about 2% to about15%, about 5% to about 15%, or about 15% to about 20%© by weight of thesecond composition. In certain embodiments, a humectant may be presentin the second composition in an amount of about 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% byweight of the second composition or any range and/or individual valuetherein.

Exemplary water repellants for the second composition include, but arenot limited to, silicones, such as cyclomethicone, dimethicone,simethicone, C26-28 alkyl dimethicone, C26-28 alkyl methicone,polyphenylsisquioxane, trimethylsiloxysilicate and crosspolymers ofcyclopentasiloxane and dimethicone/vinyltrimethylsiloxysilicate, andblends thereof. In some embodiments, a second composition may comprisecyclomethicone. A water repellant may be present in the secondcomposition in an amount of about 0.5% to about 15% by weight of thesecond composition or any range and/or individual value therein, suchas, but not limited to, about 0.5% to about 10%, about 1% to about 5%,or about 2% to about 5% by weight of the second composition. In certainembodiments, a water repellant may be present in the second compositionin an amount of about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, or 15% by weight of the second composition or anyrange and/or individual value therein.

Accordingly, a composition of the present invention may comprise atleast one polyhydric alcohol, a first viscosity increasing agent, atleast one preservative, at least one buffering agent, water, a secondviscosity increasing agent, at least one organic solvent, at least onehumectant, and optionally a water repelling agent.

A composition of the present invention (e.g., a first composition of thepresent invention and a second composition) may be buffered to a pH ofabout 3 to about 11, such as, but not limited to, about 3 to about 9.5or about 3 to about 8. In some embodiments, a composition of the presentinvention may have a pH of 9.5 or greater. In certain embodiments, thecomposition of the present invention comprises at least one API, suchas, but not limited to, a nitric oxide-releasing active pharmaceuticalingredient. In some embodiments, a composition of the present inventionmay comprise a second composition comprising a nitric oxide-releasingactive pharmaceutical ingredient.

In some embodiments, a composition of the present invention comprises asecond composition, wherein the second composition comprises a moisturesensitive API. The second composition may stably store the moisturesensitive API. In some embodiments, the moisture sensitive API maycomprise an NO-releasing API, such as, but not limited to adiazeniumdiolate modified macromolecule.

“Nitric oxide releasing active pharmaceutical ingredient” and “NOreleasing API,” as used herein, refer to a compound or other compositionthat provides nitric oxide to the skin of a subject, but is not gaseousnitric oxide. In some embodiments, the NO releasing API includes anitric oxide-releasing compound, hereinafter referred to as a“NO-releasing compound.” An NO-releasing compound includes at least oneNO donor, which is a functional group that may release nitric oxideunder certain conditions. In some embodiments, the at least one NO donorof an NO-releasing compound releases NO when in contact with acomposition of the present invention. In certain embodiments, acomposition of the present invention modulates the amount of NO releasedfrom an NO-releasing compound and/or the rate of NO released from anNO-releasing compound. In some embodiments, a composition of the presentinvention increases the amount of NO released from an NO-releasingcompound and/or the rate of NO released from an NO-releasing compound.

Any suitable NO-releasing compound may be used. In some embodiments, theNO-releasing compound includes a small molecule compound that includesan NO donor group. “Small molecule compound” as used herein is definedas a compound having a molecular weight of less than 500 daltons, andincludes organic and/or inorganic small molecule compounds. In someembodiments, the NO-releasing compound includes a macromolecule thatincludes an NO donor group. A “macromolecule” is defined herein as anycompound that has a molecular weight of 500 daltons or greater. Anysuitable macromolecule may be used, including crosslinked ornon-crosslinked polymers, dendrimers, metallic compounds, organometalliccompounds, inorganic-based compounds, and other macromolecularscaffolds. In some embodiments, the macromolecule has a nominal diameterranging from about 0.1 nm to about 100 μm and may comprise theaggregation of two or more macromolecules, whereby the macromolecularstructure is further modified with an NO donor group.

In some embodiments, the NO-releasing compound includes adiazeniumdiolate functional group as an NO donor. The diazeniumdiolatefunctional group may produce nitric oxide under certain conditions, suchas upon exposure to water. As another example, in some embodiments, theNO-releasing compound includes a nitrosothiol functional group as the NOdonor. The NO donor may produce nitric oxide under certain conditions,such as upon exposure to light. Examples of other NO donor groupsinclude nitrosamine, hydroxyl nitrosamine, hydroxyl amine andhydroxyurea. Any suitable combination of NO donors and/or NO-releasingcompounds may also be used in a second composition as described herein.Additionally, the NO donor may be incorporated into or onto the smallmolecule or macromolecule through covalent and/or non-covalentinteractions.

An NO-releasing macromolecule may be in the form of an NO-releasingparticle, such as those described in U.S. Application Publication No.2009/0214618, the disclosure of which is incorporated by referenceherein in its entirety. Other non-limiting examples of NO-releasingcompounds include NO-releasing zeolites as described in United StatesPatent Publication Nos. 2006/0269620 or 2010/0331968; NO-releasing metalorganic frameworks (MOFs) as described in United States PatentApplication Publication Nos. 2010/0239512 or 2011/0052650; NO-releasingmulti-donor compounds as described in International Application No.PCT/US2012/052350 entitled “Tunable Nitric Oxide-ReleasingMacromolecules Having Multiple Nitric Oxide Donor Structures”;NO-releasing dendrimers or metal structures as described in U.S.Publication No. 2009/0214618; nitric oxide releasing coatings asdescribed in U.S. Publication No. 2011/0086234; and compounds asdescribed in U.S. Publication No. 2010/0098733. The disclosures of eachof the references in this paragraph are incorporated herein by referencein their entirety. Additionally, NO-releasing macromolecules may befabricated as described in International Application No.PCT/US2012/022048 entitled “Temperature Controlled Sol-GelCo-Condensation” filed Jan. 20, 2012, the disclosure of which isincorporated herein by reference in its entirety.

As an example, in some embodiments of the present invention, a nitricoxide-releasing active pharmaceutical ingredient may include NO-loadedprecipitated silica. The NO-loaded precipitated silica may be formedfrom nitric oxide donor modified silane monomers into a co-condensedsiloxane network. In one embodiment of the present invention, the nitricoxide donor may be an N-diazeniumdiolate. In some embodiments of thepresent invention, the nitric oxide-releasing active pharmaceuticalingredient may comprise, consist essentially of, or consist of aco-condensed siloxane network comprising a diazeniumdiolate (e.g., aN-diazeniumdiolate).

In some embodiments, the nitric oxide donor may be formed from anaminoalkoxysilane by a pre-charging method, and the co-condensedsiloxane network may be synthesized from the condensation of a silanemixture that includes an alkoxysilane and the aminoalkoxysilane to forma nitric oxide donor modified co-condensed siloxane network. As usedherein, the “pre-charging method” means that aminoalkoxysilane is“pretreated” or “precharged” with nitric oxide prior to theco-condensation with alkoxysilane. In some embodiments, the prechargingnitric oxide may be accomplished by chemical methods. In anotherembodiment, the “pre-charging” method may be used to create co-condensedsiloxane networks and materials more densely functionalized withNO-donors. In some embodiments of the present invention, the nitricoxide-releasing active pharmaceutical ingredient may comprise, consistessentially of, or consist of a co-condensed silica network synthesizedfrom the condensation of a silane mixture comprising an alkoxysilane andat least one aminoalkoxysilane having an amine substituted by adiazeniumdiolate (e.g., a N-diazeniumdiolate).

The co-condensed siloxane network may be silica particles with a uniformsize, a collection of silica particles with a variety of size, amorphoussilica, a fumed silica, a nanocrystalline silica, ceramic silica,colloidal silica, a silica coating, a silica film, organically modifiedsilica, mesoporous silica, silica gel, bioactive glass, or any suitableform or state of silica.

In some embodiments, the alkoxysilane is a tetraalkoxysilane having theformula Si(OR)4, wherein R is an alkyl group. The R groups may be thesame or different. In some embodiments the tetraalkoxysilane is selectedas tetramethyl orthosilicate (TMOS) or tetraethyl orthosilicate (TEOS).In some embodiments, the aminoalkoxysilane has the formula:R″—(NH—R′)n-Si(OR)3, wherein R is alkyl, R′ is alkylene, branchedalkylene, or aralkylene, n is 1 or 2, and R″ is selected from the groupconsisting of alkyl, cycloalkyl, aryl, and alkylamine.

In some embodiments, the aminoalkoxysilane may be selected fromN-(6-aminohexyl)aminopropyltrimethoxysilane (AHAP3);N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEAP 3);(3-trimethoxysilylpropyl)di-ethylenetriamine (DET3);(aminoethylaminomethyl)phenethyltrimethoxysilane (AEMP3);[3-(methylamino)propyl]trimethoxysilane (MAP3);N-butylamino-propyltrimethoxysilane(n-BAP3);t-butylamino-propyltrimethoxysilane(t-BAP3);N-ethylaminoisobutyltrimethoxysilane (EAiB3);N-phenylamino-propyltrimethoxysilane (PAP3); andN-cyclohexylaminopropyltrimethoxysilane (cHAP3).

In some embodiments, the aminoalkoxysilane has the formula:NH[R′-Si(OR)3]2, wherein R is alkyl and R′ is alkylene. In someembodiments, the aminoalkoxysilane may be selected frombis(3-triethoxysilylpropyl)amine, bis-[3-(trimethoxysilyl)propyl]amineand bis-[(3-trimethoxysilyl)propyl]ethylenediamine.

In some embodiments, as described herein above, the aminoalkoxysilane isprecharged for NO-release and the amino group is substituted by adiazeniumdiolate. Therefore, in some embodiments, the aminoalkoxysilanehas the formula: R″—N(NONO—X+)—R—Si(OR)3, wherein R is alkyl, R′ isalkylene or aralkylene, R″ is alkyl or alkylamine, and X+ is a cationselected from the group consisting of Na+, K+ and Li+.

The composition of the siloxane network, (e.g., amount or the chemicalcomposition of the aminoalkoxysilane) and the nitric oxide chargingconditions (e.g., the solvent and base) may be varied to optimize theamount and duration of nitric oxide release. Thus, in some embodiments,the composition of the silica particles may be modified to regulate thehalf-life of NO release from silica particles.

In another embodiment, the amino group of aminoalkoxysilane issubstituted with a diazeniumdiolate, and the aminoalkoxysilane having aformula of R″—N(NONO—X+)—R′—Si(OR)3, wherein: R is alkyl, R′ is alkyleneor aralkylene, R″ is alkyl or alkylamine, and X+ is a cation selectedfrom the group consisting of Na+ and K+.

In certain embodiments, the NO-releasing API may comprise a co-condensedsilica network comprising diazeniumdiolated aminoethylaminopropyltrimethoxy silane (AEAP3) and tetra methyl orthosilicate (TMOS) and/or aco-condensed silica network comprising diazeniumdiolatedaminoethylaminopropyl trimethoxy silane (AEAP3) and tetraethylorthosilicate (TEOS). In some embodiments, the NO-releasing API maycomprise a co-condensed silica network comprising diazeniumdiolatedmethylaminopropyl trimethoxysilane (MAP3) and tetra methyl orthosilicate(TMOS) and/or a co-condensed silica network comprising diazeniumdiolatedmethylaminopropyl trimethoxysilane (MAP3) and tetraethyl orthosilicate(TEOS).

In some embodiments of the invention, the particle size of aNO-releasing API may be in a range of about 20 nm to about 20 μm or anyrange therein, such as, but not limited to, about 100 nm to about 20 μmor about 1 μm to about 20 μm. The particle size may be tailored tominimize or prevent toxicity and/or penetration through the epidermis(or compromised dermis) and into the blood vessels. In particularembodiments, the particle size is distributed around a mean particlesize of less than 20 μm, or any range therein, and the size may allowthe particle to enter a follicle. In some embodiments, a NO-releasingAPI may have a particle size that is distributed around a mean particlesize of about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2, or 1 μm. In further embodiments, a NO-releasing API may have aparticle size that is distributed around a mean particle size of lessthan 10 μm, or any range therein, such as, but not limited to about 2 μmto about 10 μm or about 4 μm to about 8 μm. In other embodiments, theparticle size may be distributed around a mean particle size of greaterthan 20 μm, or any range therein, and the size may prevent the particlefrom entering the follicle. In still further embodiments, a mixture ofparticles with mean particle sizes distributed around two or more meanparticle sizes may be provided. A NO-releasing API may be micronized(e.g., ball and/or jet milled). Methods for providing a desired particlesize and/or micronization include, but are not limited to, thosedescribed in U.S. Patent Application Publication No. 2013/0310533, whichis incorporated herein by reference in its entirety.

A nitric oxide-releasing active pharmaceutical ingredient may be presentin a composition of the present invention in an amount of about 0.5% toabout 10% by weight of the composition, such as, but not limited to,about 1% to about 8% or about 2% to about 6%. In certain embodiments, anitric oxide-releasing active pharmaceutical ingredient may be presentin a composition of the present invention in an amount of about 0.5%,1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. A composition of the presentinvention may comprise a nitric oxide-releasing active pharmaceuticalcomposition and may store andlor release nitric oxide in an amount ofabout 0.05% to about 3% by weight of composition, such as, but notlimited to, about 0.15% to about 2%, about 0.15% to about 1%, about 0.3%to about 1.2%. In certain embodiments, a composition of the presentinvention may comprise a nitric oxide-releasing active phaimaceuticaland may store and/or release nitric oxide in an amount of about 0.15%,0.3%, 0.6%, 0.9%, 1%, 1.2%, 1.5%, 1,75%, 2%, 2.25%, 2.5%, 2.75%, or 3%.[00981 In some embodiments, a first composition of the present inventionmay increase the amount of NO released from a composition (e.g., asecond composition comprising a NO-releasing API) compared to the amountof NO released from the composition in the absence of a firstcomposition of the present invention over the same period of time. Incertain embodiments, a first composition of the present invention mayincrease the amount of NO released from a composition of the presentinvention comprising the first composition and a NO-releasing API by atleast about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90, or more, or anyrange and/or individual value therein compared to the amount of NOreleased in the absence of a first composition of the present inventionover the same period of time. Therefore, a composition of the presentinvention (e.g., a composition comprising a first composition of thepresent invention and a second composition comprising a NO-releasingAPI) may release about 1.5 to about 100 times more NO than the amount ofNO released in the absence of a first composition of the presentinvention (e.g., the second composition alone) over the same period oftime or any range and/or individual value therein, such as, but notlimited to between about 2 and 10 times more NO or between about 5 andabout 50 times more NO.

For example, the effect a composition of the present invention may haveon the amount of NO released can be seen in FIGS. 7 and 8. FIG. 7 showsthe in vitro nitric oxide release profile for a 2% Nitricil™ NVN1 Gelhaving a formulation as set forth in Table 1 over time. FIG. 8 shows thein vitro nitric oxide release profiles for the 2% Nitricil™NVN1 Gel anda 6% and 12% Nitricil™ NVN1 Gel having formulations as set forth inTable 1 upon mixing with a first composition of the present invention atpH 4 having a formulation as set forth in Table 2 in a 1:3 ratio(gel:first composition) over time. As can be seen from FIGS. 7 and 8 thecumulative NO release for the 2% Nitricil™ NVN1 Gel increased when incontact with a first composition of the present invention.

TABLE 1 Composition of the 2%, 6%, and 12% Nitricil ™ NVN1 gels. % w/wGel Component Vehicle 2% 6% 12% Isopropyl alcohol 85.5 83.5 80.5 74.5Hexylene glycol 10.0 10.0 10.0 10.0 Cyclomethicone 2.5 2.5 2.5 2.5Hydroxypropyl cellulose 2.0 2.0 1.0 1.0 Nitricil ™ NVN1 0 2.0 6.0 12.0

TABLE 2 Composition of the first composition with a pH of 4. Component %w/w Purified water 89.1 Glycerin 10.0 Carbopol ® 974P 0.5 Sorbic acid0.2 Trolamine 0.2

A composition of the present invention may comprise a first compositionof the present invention and a second composition as described herein.As those of skill in the art will recognize, the amount or concentrationof individual components in a composition of the present invention mayvary depending on the amount of the first composition and secondcomposition present in the composition (e.g., the ratio of the firstcomposition and second composition present in the composition). In someembodiments, the ratio of a first composition of the present inventionto a second composition in a composition of the present invention may beabout 5:1 or less, in further embodiments, about 4:1 or less, about 3:1or less, about 2:1 or less, about 1:1 or less, about 0.5:1 or less, orabout 0.2:1 or less. In particular embodiments, the ratio may be about3:1. In further embodiments, the ratio may be about 1:1.

In some embodiments, a composition of the present invention maycomprise, consist essentially of, or consist of a polyhydric alcohol inan amount of about 1% to about 10% by weight of the composition, a firstviscosity increasing agent in an amount of about 0.01% to about 3% byweight of the composition, water in an amount of about 30% to about 50%by weight of the composition, a second viscosity increasing agent in anamount of about 0.01% to about 10% by weight of the composition, anorganic solvent in an amount of about 30% to about 50% by weight of thecomposition, a humectant present in the second composition in an amountof about 2% to about 10% by weight of the composition, a water repellingagent in an amount of about 0.1% to about 10% by weight of thecomposition, an NO-releasing API in an amount of about 0.5% to about 10%by weight of the composition, optionally a buffering agent in an amountof about 0.001% to about 1% by weight of the composition, optionally apreservative in an amount of about 0.001% to about 1% by weight of thecomposition, and optionally a neutralizing agent. The neutralizing agentmay be present in an amount sufficient to provide the first part of thecomposition with a pH of about 3 to about 8. The composition may have apH of less than about 11, such as, but not limited to, less than about9.5, less than about 7, or less than about 6. The first and secondviscosity increasing agents may be the same and/or different. In certainembodiments, the first viscosity increasing agent may be acarboxypolymethylene and the second viscosity increasing agent may be acellulose, such as, but not limited to, hydroxypropyl cellulose. In someembodiments, the composition may be cosmetically elegant.

In some embodiments, a composition of the present invention maycomprise, consist essentially of, or consist of a polyhydric alcohol inan amount of about 2% to about 7% by weight of the composition, a firstviscosity increasing agent in an amount of about 0.1% to about 1% byweight of the composition, water in an amount of about 40% to about 45%by weight of the composition, a second viscosity increasing agent in anamount of about 0.1% to about 1% by weight of the composition, anorganic solvent in an amount of about 35% to about 45% by weight of thecomposition, a humectant present in the second composition in an amountof about 2% to about 7% by weight of the composition, a water repellingagent in an amount of about 0.1% to about 5% by weight of thecomposition, an NO-releasing API in an amount of about 0.5% to about 10%by weight of the composition, optionally a buffering agent in an amountof about 0.01% to about 0.2% by weight of the composition, optionally apreservative in an amount of about 0.01% to about 0.3% by weight of thecomposition, and optionally a neutralizing agent. The neutralizing maybe present in an amount sufficient to provide the first part of thecomposition with a pH of about 4 or about 6. The composition may have apH of less than about 11, such as, but not limited to, less than about9.5, less than about 7, or less than about 6_(—) The first and secondviscosity increasing agents may be the same and/or different. In certainembodiments, the first viscosity increasing agent may be acarboxypolymethylene and the second viscosity increasing agent may be acellulose, such as, but not limited to, hydroxypropyl cellulose. In someembodiments, the composition may be cosmetically elegant. In certainembodiments, the composition may comprise a composition as set forth inTable 13.

A composition of the present invention may comprise at least twodifferent viscosity increasing agents. One viscosity increasing agentmay be present in the first part of a composition of the presentinvention and the other viscosity increasing agent may be present in thesecond part of the composition. In some embodiments, a composition ofthe present invention comprises a carboxypolymethylene and a cellulose,such as, but not limited to, hydroxypropyl cellulose.Carboxypolymethylene may be present in a first composition of thepresent invention and the cellulose may be present in a secondcomposition, which may be combined to form a composition of the presentinvention. A composition of the present invention comprising at leasttwo different viscosity increasing agents may provide a cosmeticallyelegant composition comprising an API, such as, but not limited to, aparticulate API and/or an insoluble API (e.g., an aqueous and/ormoisture insoluble API, such as, for example, benzoyl peroxide).

A composition of the present invention may provide a structure suitablefor suspending an API, such as, but not limited to, a particulate APIand/or an insoluble API. In some embodiments, a composition of thepresent invention may encapsulate an API, such as, but not limited to, aparticulate API and/or an insoluble API. For example, a composition ofthe present invention may encapsulate an API in at least one viscosityincreasing agent and/or may provide a structure suitable forencapsulating an API. A composition of the present invention may preventand/or reduce agglomeration of an API in the composition. While notwishing to be bound to any particular theory, a first composition of thepresent invention may provide means for suspending and/or encapsulatingan API and/or for preventing and/or reducing agglomeration of an API inthe composition. For example, a first composition of the presentinvention may provide a structure (e.g., a gel matrix) suitable forsuspending and/or encapsulating an API in a composition of the presentinvention and/or for preventing and/or reducing agglomeration of an APIin a composition of the present invention.

A composition of the present invention may comprise an API, acarboxypolymethylene, and a cellulose and may be a cosmetically elegantcomposition. The composition may not be gritty and/or may have a reducedgrittiness compared to the API in the absence of a composition of thepresent invention. The composition may not be tacky (i.e., sticky)and/or may have a reduced tackiness (i.e., stickiness) compared to theAPI in the absence of a composition of the present invention. Thecomposition may have a reduced and/or increased stiffness (i.e.,hardness) and/or may have an increased homogeneity compared to the APIin the absence of a composition of the present invention. In someembodiments, a composition of the present invention may comprise an APIand may be a cosmetically elegant, homogeneous composition. While notwishing to be bound to any particular theory, a first composition of thepresent invention may provide means for providing a cosmeticallyelegant, homogeneous composition. For example, a first composition ofthe present invention may provide a structure (e.g., a gel matrix)suitable for providing a cosmetically elegant, homogeneous composition.

The first part and/or composition of a composition of the presentinvention and the second part and/or composition of the composition ofthe present invention may be contacted (e.g., mixed, stirred, blended,homogenized, and the like) during the process of compounding thecomposition. The composition of the present invention may then be storedwith the first part and/or composition and the second part and/orcomposition combined (e.g., in the same vessel and/or container).

A composition of the present invention may comprise an API that isdifficult to formulate, such as, but not limited to, an API that isdifficult to formulate for a topical composition. For example, the APImay be a particulate API, an insoluble API (e.g., an aqueous and/ormoisture insoluble API), a thermally unstable API, and/or aphotosensitive API.

According to embodiments of the present invention, the preparation oftwo separate parts for a composition of the present invention mayprovide an improved composition. A composition of the present inventioncomprising at least two parts may allow for an API (e.g., an API that isdifficult to formulate) to be prepared in one part and later combinedwith the second part to prepare a cosmetically elegant compositionand/or a composition comprising a more stable API compared to the sameAPI prepared in a composition formed with one part and/or in the absenceof a composition of the present invention. The API may be more stable inthat the API could have a greater activity compared to its activity inthe absence of a composition of the present invention and/or the API maybe stored for a longer period of time compared to its storage in theabsence of a composition of the present invention. In some embodiments,a composition of the present invention comprising an API (e.g., an APIthat is difficult to formulate) may provide a more cosmetically elegantcomposition compared to the API in a composition in the absence of acomposition of the present invention. For example, a composition of thepresent invention comprising a particulate API and/or an insoluble APImay provide a less gritty composition and thereby a more cosmeticallyelegant composition.

According to embodiments of the present invention, a kit may beprovided. In some embodiments, a kit of the present invention maycomprise a first composition of the present invention and a secondcomposition as described herein. The first composition may be ahydrogel. The first composition may comprise at least one polyhydricalcohol present in an amount of about 1% to about 30% by weight of thefirst composition, at least one viscosity increasing agent present in anamount of about 0.1% to about 5% by weight of the first composition, andwater present in an amount of about 70% to about 99% by weight of thefirst composition. The second composition may comprise an API, such as,but not limited to, an NO releasing API. In some embodiments, the secondcomposition may comprise at least one viscosity increasing agent presentin the second composition in an amount of about 0.5% to about 30% byweight of the second composition, at least one organic solvent presentin the second composition in an amount of about 50% to about 90 byweight of the second composition, and at least one humectant present inthe second composition in an amount of about 2% to about 20% by weightof the second composition. In particular embodiments, the secondcomposition may comprise an anhydrous alcohol gel containing a nitricoxide-releasing polysiloxane macromolecule as described in InternationalApplication Publication No. WO 2013/006608.

In some embodiments, a kit of the present invention comprises an aqueouscomposition, an organic composition, and an API that may be stableand/or soluble in the aqueous composition and/or the organiccomposition. A kit of the present invention may be configured to mix thetwo compositions upon dispensing and/or for application to a subjectand/or may be configured to provide a combined composition with,increased performance and/or activity of the API compared to theperformance and/or activity of the API in the absence of one of thecompositions and/or the combined composition.

A kit of the present invention may separately store a first compositionof the present invention and a second composition. In some embodiments,a kit of the present invention may contact the first composition andsecond composition, such as, but not limited to, by mixing thecompositions, prior to application to a subject.

A first composition of the present invention (e.g., a hydrogel of thepresent invention) and a second composition may be mixed together andthen applied to the skin of a subject. In other embodiments, a secondcomposition may be applied to the skin of a subject and then a firstcomposition may be applied over the second composition or vice versa. Insome embodiments, the ratio of a first composition to a secondcomposition, which may be applied to a subject, may be about 5:1 orless, in further embodiments, about 4:1 or less, about 3:1 or less,about 2:1 or less or about 1:1. In particular embodiments, the ratio maybe about 3:1. In further embodiments, the ratio may be about 1:1.

Providing a first composition and a second composition that are combinedupon and/or during application to the skin of a subject may allow for alonger shelf life of a kit of the present invention than if thecompositions were stored mixed together in the kit. For example, theformulation and loading of API in the second composition may provide astable product with a long shelf life. Thus, for example, pH and watercontent of the second composition may be adjusted to reduce or minimizerelease of a water-activated API so as to provide a composition that isstable at room temperature. The first composition may then be combinedwith the second composition to adjust the combined pH and provide waterto activate the API. The second composition may be combined with thefirst composition in differing ratios to provide a desired release, pHand/or dose in the combined composition. Such an approach may allow fora single manufacturing process to be utilized for production of a morecomplex and costly second composition and then particular productsdefined by the composition and/or quantity of the first composition withwhich the second composition is mixed.

As will be appreciated by those of skill in the art in light of thepresent disclosure, a first composition of the present invention (e.g.,a hydrogel of the present invention) may provide means for adjusting thepH of a pharmaceutical composition as well as means for activating anAPI of a pharmaceutical composition. In particular embodiments, a firstcomposition of the present invention may provide means for reducing thepH of an anhydrous pharmaceutical composition comprising adiazeniumdiolate modified co-condensed polysiloxane macromolecule. Infurther embodiments, a first composition of the present invention mayprovide means for releasing nitric oxide from an anhydrouspharmaceutical composition comprising a diazeniumdiolate modifiedco-condensed polysiloxane macromolecule.

According to some embodiments, a method of the present invention maycomprise administering a composition of the present invention (e.g., afirst composition of the present invention and a second composition asdescribed herein) and/or first composition of the present invention(e.g., a hydrogel of the present invention) to the skin of a subject. Incertain embodiments, the composition and/or first composition may betopically administered. The composition may comprise at least one API,such as, but not limited to, a nitric oxide-releasing activepharmaceutical ingredient; a second viscosity increasing agent; at leastone organic solvent; at least one humectant; at least one polyhydricalcohol; a first viscosity increasing agent; at least one preservative;and water. A method of the present invention may comprise forming anadmixture prior to and/or during administration of a composition of thepresent invention. An admixture may be prepared by mixing or combining acomposition comprising at least one API, such as, but not limited to, anitric oxide-releasing active pharmaceutical ingredient; a secondviscosity increasing agent; at least one organic solvent; and at leastone humectant, and a composition comprising at least one polyhydricalcohol; a first viscosity increasing agent; at least one preservative;and water.

A method of the present invention may comprise topically applying afirst composition of the present invention to the skin of a subject incombination and/or admixture with a second composition. The secondcomposition may comprise a nitric oxide-releasing active pharmaceuticalingredient.

In some embodiments, a method of the present invention comprisesdelivering a therapeutically effective amount of a composition of thepresent invention to the skin of a subject. As used herein, the term“therapeutically effective amount” refers to an amount of a compositionof the present invention that elicits a therapeutically useful responsein a subject. Those skilled in the art will appreciate that thetherapeutic effects need not be complete or curative, as long as somebenefit is provided to the subject. In some embodiments, atherapeutically effective amount of a composition of the presentinvention may include delivering a therapeutically effective amount of acomponent of the composition, such as, but not limited to, an activepharmaceutical ingredient (e.g., a nitric oxide-releasing API).Therefore, a therapeutically effective amount of nitric oxide may bedelivered and/or administered by a composition of the present invention.

The present invention finds use in both veterinary and medicalapplications. Subjects suitable to be treated with a method embodimentof the invention include, but are not limited to, avian and mammaliansubjects. Mammals of the present invention include, but are not limitedto, canines, felines, bovines, caprines, equines, ovines, porcines,rodents (e.g. rats and mice), lagomorphs, primates (e.g., simians andhumans), non-human primates (e.g., monkeys, baboons, chimpanzees,gorillas), and the like, and mammals in utero. Any mammalian subject inneed of being treated according to the present invention is suitable.Human subjects of both genders and at any stage of development (i.e.,neonate, infant, juvenile, adolescent, adult) may be treated accordingto the present invention. In some embodiments of the present invention,the subject is a mammal and in certain embodiments the subject is ahuman. Human subjects include both males and females of all agesincluding fetal, neonatal, infant, juvenile, adolescent, adult, andgeriatric subjects as well as pregnant subjects. In particularembodiments of the present invention, the subject is a human adolescentand/or adult.

Illustrative avians according to the present invention include chickens,ducks, turkeys, geese, quail, pheasant, ratites (e.g., ostrich) anddomesticated birds (e.g., parrots and canaries), and birds in ovo.

The methods of the present invention may also be carried out on animalsubjects, particularly mammalian subjects such as mice, rats, dogs,cats, livestock and horses for veterinary purposes, and/or for drugscreening and drug development purposes.

In particular embodiments of the present invention, the subject is “inneed of” a method of the present invention, e.g., the subject has beendiagnosed with, is at risk for, and/or is believed to have a disease ordisorder that may be treated using a method of the present invention. Insome embodiments, the subject has a skin disorder, such as, but notlimited to, acne, androgenetic alopecia, atopic dermatitis, seborrheicdermatitis, tinea infections, candida infections, bacterial infections,verruca vulgaris, and/or psoriasis. In some embodiments of the presentinvention, the subject has an inflammatory skin condition or disorderand/or infection (e.g., impetigo, leishmaniasis, etc.). In someembodiments, a composition of the present invention may be used to treatacne vulgaris. According to some embodiments, a composition and/ormethod of the present invention may reduce P. acnes counts, inflammatorylesions, and/or noninflammatory lesions in a subject. In someembodiments, a composition and/or method of the present invention may beused to treat any disease, disorder, and/or condition suitable fortopical administration with a composition comprising an NO-releasing APIand an alcohol excipient, such as, but not limited to, alopecia (e.g.,androgenetic alopecia) and/or a wart (e.g., a common, plantar, flat,filiform, genital, mosaic, and/or periungual wart).

“Treat,” “treating” or “treatment of” (and grammatical variationsthereof) as used herein refer to any type of treatment that imparts abenefit to a subject and may mean that the severity of the subject'scondition is reduced, at least partially improved or ameliorated and/orthat some alleviation, mitigation or decrease in at least one clinicalsymptom is achieved and/or there is a delay in the progression of thedisease, disorder, and/or condition. In particular embodiments, theseverity of a skin disorder (e.g., acne) may be reduced in a subjectcompared to the severity of the skin disorder in the absence of a methodof the present invention. In other embodiments, a method of the presentinvention may prevent and/or treat against infection.

A composition of the present invention may be applied topically to anyportion of a subject's skin. However, in some embodiments, the subject'sface is treated by a method described herein. Furthermore, in someembodiments, the subject's trunk is treated by a method describedherein. In some embodiments, the subject's hand(s), finger(s), foot,feet, toe(s), and/or genital(s) are treated by a method describedherein.

According to some embodiments of the present invention, a method oftreating acne vulgaris may be provided, the method comprising topicallyapplying a composition of the present invention to the skin of asubject. A therapeutically effective amount of the composition may beapplied. In some embodiments, the composition may comprise a nitricoxide-releasing active pharmaceutical ingredient in an amount of about0.5% to about 10% by weight of the composition, such as, but not limitedto, about 1% to about 8% or about 2% to about 6%. In certainembodiments, the composition may comprise a nitric oxide-releasingactive pharmaceutical ingredient in an amount of about 0.5%, 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, or 10%. The composition may store and/or releasenitric oxide in an amount of about 0.05% to about 3% by weight ofcomposition, such as, but not limited to, about 0.15% to about 2%, about0.15% to about 1%, about 0.3% to about 1.2%. In certain embodiments, thecomposition may store and/or release nitric oxide in an amount of about0.15%, 0.3%, 0.6%, 0.9%, 1%, 1.2%, 1.5%, 1.75%, 2%, 2.25%, 2.5%, 2.75%,or 3%.

According to further embodiments of the present invention, a method ofreducing inflammatory and/or noninflammatory lesions in a subject may beprovided comprising topically applying a composition of the presentinvention to the skin of the subject. A therapeutically effective amountof the composition may be applied. In some embodiments, the compositionmay comprise a nitric oxide-releasing active pharmaceutical ingredientin an amount of about 0.5% to about 10% by weight of the composition,such as, but not limited to, about 1% to about 8% or about 2% to about6%. In certain embodiments, the composition may comprise a nitricoxide-releasing active pharmaceutical ingredient in an amount of about0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. The composition maystore and/or release nitric oxide in an amount of about 0.05% to about3% by weight of composition, such as, but not limited to, about 0.15% toabout 2%, about 0.15% to about 1%, about 0.3% to about 1.2%. In certainembodiments, the composition may store and/or release nitric oxide in anamount of about 0.15%, 0.3%, 0.6%, 0.9%, 1%, 1.2%, 1.5%, 1.75%, 2%,2.25%, 2.5%, 2.75%, or 3%.

The method of reducing inflammatory and/or noninflammatory lesions maycomprise topically applying a combined composition of the presentinvention. In some embodiments, the method may reduce inflammatoryand/or noninflammatory lesions by about 10% or greater, such as, but notlimited to, about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or more, over a defined period of timecompared to a subject who did not apply a composition of the presentinvention over the same time period. In certain embodiments, the methodmay reduce inflammatory and/or noninflammatory lesions in a subjectcompared to a subject who did not apply a composition comprising anitric oxide-releasing active pharmaceutical ingredient over the sameperiod of time.

In some embodiments, a method of reducing inflammatory and/ornoninflammatory lesions in a subject may be provided comprisingtopically applying a first composition of the present invention to theskin of the subject. The first composition may not comprise an API, suchas, but not limited to, an NO-releasing API. A therapeutically effectiveamount of the first composition may be applied. The method of reducinginflammatory and/or noninflammatory lesions comprising topicallyapplying a first composition of the present invention may reduceinflammatory and/or noninflammatory lesions by about 10% or greater,such as, but not limited to, about 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more, over adefined period of time compared to a subject who did not apply a firstcomposition of the present invention over the same time period.

In some embodiments, a method of the present invention may comprisetopically applying a composition of the present invention comprising anitric oxide-releasing active pharmaceutical ingredient, wherein themethod may reduce inflammatory and/or noninflammatory lesions by about10% or greater, such as, but not limited to, about 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, ormore, over a defined period of time compared to a subject who appliedsubstantially the same composition without the nitric oxide-releasingactive pharmaceutical ingredient.

In certain embodiments, the subject may see a reduction in inflammatoryand/or noninflammatory lesions within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, or more week(s). In some embodiments, the method may reduceinflammatory and/or noninflammatory lesions in the skin of the subjectwith 12 weeks or less, in some embodiments, within 8 weeks or less, andin further embodiments, within 4 weeks or less.

In some embodiments, a method of the present invention may reduce P.acnes counts in a subject administered and/or topically applying acomposition of the present invention. In certain embodiments, a methodof the present invention may reduce P. acnes counts by about 10% orgreater, such as, but not limited to, about 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more overa defined period of time compared to a subject who did not apply acomposition of the present invention over the same time period. In someembodiments, the method may reduce P. acnes counts in a subject comparedto a subject who did not apply a composition comprising a nitricoxide-releasing active pharmaceutical ingredient over the same period oftime.

In certain embodiments, a reduction in P. acnes counts may occur within1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or more week(s). In someembodiments, a method of the present invention may reduce P. acnescounts in the skin of the subject with 12 weeks or less, in someembodiments, within 8 weeks or less, and in further embodiments, within4 weeks or less.

The present invention is explained in greater detail in the followingnon-limiting Examples.

EXAMPLES Example 1

Unbuffered hydrogel formulations with pH values ranging between 3 and 7were developed. Several pH 6 hydrogel formulations were manufacturedwith varying levels of carbomer Carbopol® 974P to investigate theeffects on viscosity and gel rheology. The effect of preservatives suchas sorbic acid, benzoic and parabens were also investigated on theunbuffered hydrogel rheology and viscosity. The manufactured hydrogelformulations were used in measuring the admixture pH values (Example 2)with Nitricil™ NVN1 topical gel formulations comprising isopropylalcohol (IPA) and varying strengths of Nitricil™ NVN1, and inestablishing in vitro nitric oxide (NO) release kinetics (Example 3). Abuffered pH 4 hydrogel with 0.1% w/w citric acid using Carpobol® 980Pand a buffered pH 6 hydrogel using Carbopol® ETD 2020NF polymer with0.2% w/w 0.1M phosphate buffer were also formulated.

For all formulations provided in Tables 3 and 4, United StatesPharmacopeia (USP) grade water and anhydrous glycerol were mixed ineither a 0.5-L or 2-L glass beaker using an IKA overhead mixer atambient temperature. For the hydrogel formulations containing apreservative such as sorbic acid, benzoic acid, and methyl- andpropyl-paraben, the preservative was added to the water and glycerolsolution and heated using a hot plate to 70° C. for complete dissolutionto occur. Once dissolution occurred, the solution was cooled down toambient temperature. The next step in each experiment was to slowlytransfer a Carbopol® polymer to the beaker with constant agitation usinga combination of overhead stirring and homogenization using the IKA T-18mixer at speeds of 3-4 for 20-30 seconds. A clear solution folioed after20 minutes indicating complete polymer dissolution. While undercontinuous agitation, the pH of the un-neutralized mixture was measuredinitially and trolamine was used as a neutralizing agent in a quantitysufficient (QS) to adjust the pH to the desired value and thicken thehydrogel. Finally, once the desired pH was obtained, a final quantity ofwater was added to reach the desired target batch size. For Batch Lot112331, titanium dioxide was introduced into the hydrogel as a maskingagent.

TABLE 3 Unbuffered hydrogel formulations without a preservative HydrogelFormulation [% w/w composition] Batch lot: Batch lot: Batch lot: Batchlot: Batch lot: Batch lot: Ingredient 112333 112335 112337 112339 112345112353 Anhydrous Glycerol, 10.0 10.0 10.0 10.0 10.0 10.0 ACS CarbomerHomopolymer 1.0 0.5 0.2 0.3 0.4 0.5 Type A, NF, Carbopol ® 974P PurifiedWater, USP 86.5 86.5 86.5 86.5 85.0 85.0 Trolamine, NF QS to pH 7 QS topH 6 QS to pH 6 QS to pH 6 QS to pH 4 QS to pH 4 Purified Water, USP QSQS QS QS QS QS (adjustment)

TABLE 4 Unbuffered hydrogel formulations with a preservative. HydrogelFormulation [% w/w composition] Batch lot: Batch lot: Batch lot: Batchlot: Batch lot: Batch lot: Batch lot: Ingredient 112331 112355 112357112359 112361 112363 112365 Anhydrous Glycerol, 10.0 10.0 10.0 10.0 10.010.0 10.0 ACS Benzoic Acid, NF 0.1 — — — — — — Sorbic Acid, NF 0.1 — 0.20.1 0.2 0.1 — Methyl paraben — 0.2 — — — — 0.2 Propyl paraben — 0.05 — —— — 0.1 Carbomer Homopolymer 1.5 0.5 0.5 0.75 0.35 0.7 0.5 Type A, NF,Carbopol 974P Purified Water, USP 85.0 85.0 86.5 86.5 86.5 86.5 85.0Titanium dioxide, NF 0.05 — — — — — — Trolamine, NF QS to pH 3 QS to pH4 QS to pH 4 QS to pH 4 QS to pH 4 QS to pH 5 QS to pH 5 Purified Water,USP QS QS QS QS QS QS QS (adjustment)

The pH of un-neutralized mixtures with and without preservativescontaining anionic Carbopol® polymer 974P was approximately 2.75-3depending on the polymer concentration. The viscosity of the pH 3hydrogel was found to be very low as only a small quantity of trolaminewas added to adjust the pH to 3 units and therefore the thickeningeffects were not realized. To fully neutralize the Carbopol® 974Ppolymer a pH adjustment to 6 or 7 was necessary using trolamine.However, the viscosity of the resultant unbuffered hydrogels was veryhigh at pH 6 and 7. While not wishing to be bound to any particulartheory, for dispensing purposes, the concentration of Carbopol® 974Ppolymer may need to be reduced from 1% w/w to 0.3-0.5% w/w to lower theviscosity at pH 6 and 7. Using Carbopol® 974P polymer concentrationsless than 0.3% w/w, while not wishing to be bound to any particulartheory, may result in a hydrogel that is not viscous enough and may runoff the surface of the skin when applied.

A pH 4 unbuffered hydrogel can be formulated with 0.5% w/w Carbopol® andhave adequate viscosity and rheological properties to flow and dispensefrom a pump but also not run off the surface of the skin when applied.Table 5 shows the viscosity measurements of several unbuffered pH 4 andpH 5 hydrogels containing preservative(s) as described in Table 4.

TABLE 5 Viscosity measurements for unbuffered hydrogels with apreservative. Batch Lot Viscosity [cP] 112355 12675 112357 6961 1123598631 112361 9372 112363 18376 112365 20746

The pH 4 hydrogels with a preservative had a viscosity ranging from7000-12500 cP. The addition of preservative influenced the pH andtherefore the neutralization (thickening) process using trolamine.Without wishing to be bound to any particular theory, to account for thepresence of preservative and various concentrations of preservative, thelevel of Carbopol® polymer may need to be adjusted accordingly to obtainconsistent viscosity post neutralization with base. Increasing the pH to5 for an unbuffered hydrogel with preservative resulted in a significantincrease in viscosity to approximately 20000 cP.

With Carbopol® 974P polymer, several buffering acids such as citricacid, tartaric acid and lactic acid were used to buffer at a pH of 4,but the hydrogels would instantaneously break down into water. Carbopol®ETD 2020, NF polymer was found to not be suitable for use with bufferingagents at concentrations of 1-3% w/w.

A phosphate buffered pH 6 hydrogel was manufactured using 0.2% w/wCarbopol® ETD 2020 NF polymer in USP water and anhydrous glycerol. A 0.1M stock of potassium phosphate buffer was added at 0.2% w/w to bufferthe hydrogel at a pH of 6 units. This hydrogel was used with Nitricil™NVN1 topical gels containing IPA and various strengths of Nitricil™ NVN1to determine admixture pH in vitro (Example 2). The pH 6 phosphatebuffered hydrogel had the effect of reducing the pH by 0.5 units atseveral different Nitricil™ NVN1 concentrations ranging from 0.2% to 8%w/w.

A citric acid buffered pH 4 hydrogel containing 0.1% w/w benzoic acidand 0.1% sorbic acid with 1% w/w Carbopol® polymer 980P was successfullycompounded at a 0.5-kg scale. The composition of the buffered citricacid hydrogel is listed in Table 6. This buffered hydrogel (Batch lot:126335) was measured to have a viscosity of 7285 cP. The citric acidbuffered hydrogel manufactured was used to determine in vitro and skinsurface pH (Example 2) including establishing in vitro nitric oxiderelease profiles (Example 3).

TABLE 6 Ingredient list and composition of the citric acid buffered pH 4hydrogel. % w/w Hydrogel ingredient (Batch lot: 126335) compositionAnhydrous Glycerol, ACS 10.0 Benzoic Acid, NF 0.1 Sorbic Acid, NF 0.1Citric acid 0.1 Carbomer Homopolymer Type C, NF, 1 Carbopol 980PPurified Water, USP 70 Trolamine, NF QS to pH4 Purified Water, USP 10(adjustment A) Purified Water, USP QS (adjustment B)

Hydrogel formulations covering a range of pH values were formulated. Theinitial hydrogels formulated were unbuffered and compounded with andwithout preservatives. Preservatives such as benzoic acid, sorbic acidand parabens were used. Parabens were found to react with Nitricil™ NVN1IPA topical gels. The pH of the hydrogels was adjusted by varying thequantity of trolamine (neutralizing agent) added. To increase the pH andviscosity, the amount of neutralizing agent added was increased. At pH 6and 7, the viscosity of the unbuffered hydrogels without preservativesformed was high. In order to reduce the hydrogel viscosity, theCarbopol® polymer concentration was reduced. The addition ofpreservatives also influenced the initial pH. The amount of polymer andneutralizing agent was adjusted accordingly to reach the desired pH andobtain a viscosity that is not too low as to cause issues with runofffrom the surface of the skin and not too high such as to cause issueswith product flow and pumping from a dual chamber dispensing device.Attempts were made to manufacture a buffered pH 4 hydrogel usingCarpobol® 974P and ETD 2020 polymer with citric acid, lactic acid, andtartaric acid as buffering agents but this resulted in the polymersbreaking down into water due to rapid changes in pH.

By switching to Carbopol® 980P, which is a Homopolymer Type C and alonger chain polymer, a 0.1% w/w citric acid buffered pH 4 hydrogel withsorbic acid and benzoic acid was successfully formulated. A pH 6hydrogel that was buffered with 0.1 M phosphate buffer at 0.2% w/w wasalso successfully formulated with Carbopol¹ ETD 2020 polymer.

Example 2

A series of experiments were carried out to determine the finaladmixture pH of Nitricil™ NVN1 topical gels containing IPA and havingdifferent strengths of Nitricil™ NVN1 ranging from 0.2-12% w/w withunbuffered and buffered hydrogel formulations having a pH ranging from 4to 6 at different hydrogel to Nitricil™ NVN1 topical gel ratios rangingfrom 1:1 to 3:1 (Hydrogel to Nitricil™ NVN1 topical gel). The effect ofhydrogel pH, mixing ratio of hydrogel to Nitricil™ NVN1 IPA topical gel,and Nitricil™ NVN1 strength on the final admixture pH was investigated.The aim of the experiments was to determine whether a final admixture pHranging between 6 to 8 could be obtained.

For all in vitro pH admixture measurements, approximately 1-g quantityof Nitricil™ NVN1 Topical IPA Gel was dispensed into a tared weigh boatusing either a 1 mL plastic syringe or directly dispensed from aluminumtubes. Once approximately 1 g quantity was dispensed, the weigh boat wasre-tared. A pre-determined quantity of hydrogel ranging from 1 to 3-gwas then dispensed into the weigh boat using a 1 mL syringe. Theadmixture was then mixed using pH probe (Beckman φ350 pH meter) until asingle steady state pH measurement was recorded. All dispensing was doneon a weight basis.

A pH 6 phosphate buffered hydrogel, an unbuffered pH 4 hydrogel, and anunbuffered pH 6 hydrogel were used to determine admixture pH values asshown in Table 7.

TABLE 7 Hydrogel formulations used in the initial admixture pHdeterminations. % w/w pH 6 Ingredient pH 4 pH 6 (Buffered) PurifiedWater, USP 85.0 85.0 85.0 Glycerol, NF 10.0 10.0 10.0 CarbomerHomopolymer 1.0 0.3 — Type A, NF Carbopol 974P Carbomer Interpolymer — —0.5 Type B, NF Carbopol ETD2020NF Trolamine, NF QS to pH 4 QS to pH 6 QSto pH 6 Purified Water, USP QS QS — 0.1M Phosphate Buffer — — QS (pH6.0)

FIG. 1 shows the effect of Nitricil™ NVN1 Topical Gel strength, hydrogelpH, and hydrogel to Nitricil™ NVN1 IPA Topical Gel ratio on theadmixture pH. The admixture pH results demonstrate that in order toachieve a pH of 8 for the final admixture, Nitricil™ NVN1 Topical Gel ator below 6% w/w Nitricil™ NVN1 could be used in combination with thehydrogel pH 4 at either a 1:1 or 3:1 ratio. At strengths of 8% w/wNitricil™ NVN1 or greater, the strong buffering properties of Nitricil™NVN1 start to heavily influence the pH of the admixture over the rangeof hydrogel pH's and ratios evaluated. Significant foaming (indicatingnitric oxide release) was observed at strengths greater than 4% w/wNitricil™ NVN1 when mixing with both pH 4 and pH 6 hydrogels. This wasobserved even at high pH admixture values greater than 8 when using pH 6hydrogels. The 1:3 ratio of Nitricil™ NVN1 Topical Gel and hydrogelbuffered at pH 6 allowed the resulting pH of the admixture to bemaintained below pH 8 for Nitricil™ NVN1 Topical Gel strengths up to 2%w/w Nitricil™ NVN1. Buffering the hydrogel with phosphate at pH 6 helpedreduce the final admixture pH at both 1:1 and 1:3 mixing ratios ofhydrogel to Nitricil™ NVN1 IPA Topical Gel.

Further experiments investigated the effect of adding preservatives(e.g., parabens, sorbic acid and benzoic acid) at concentrations rangingbetween 0.1% to 0.2% w/w to hydrogel foimulations adjusted to pH 4 andpH 5. The admixture pH was measured for Nitricil™ NVN1 Topical Gelstrengths at 2% w/w and 8% w/w.

Table 8 shows the admixture pH values using both 2% and 8% w/w Nitricil™NVN1. The results in Table 8 show that the 2% w/w Nitricil™ NVN1 IPATopical Gel with the pH 4 hydrogel, with and without preservatives, at aratio of 1:1 provided a resultant admixture pH between 6 and 7.

TABLE 8 Admixture pH values for different pH hydrogels with and withoutpreservative. Nitricil ™ NVN1 Concentration [% w/w] Hydrogel pHPreservative [% w/w] Admixture pH 2 4 Methyl-and Propyl-paraben 0.256.61 (Gel mixture turns brown) 2 4 Sorbic acid 0.2 5.89 2 4 Sorbic acid0.1 6.73 2 4 No preservative — 6.78 8 4 0.2% methyl- and 0.5% propyl-0.25 9.93 paraben 8 4 Sorbic acid 0.2 10.19 8 4 Sorbic acid 0.1 10.27 84 None — 8.25 8 4 None — 10.80 2 5 Methyl-and Propyl-paraben 0.25 7.15(Gel mixture turns brown) 2 5 Sorbic acid 0.2 6.81 2 5 Sorbic acid 0.16.94

Using methyl- and propyl-paraben as preservatives resulted in theadmixture turning brown. Without wishing to be bound to any particulartheory, this could be indicative of the formation of degradationproduct(s). Using pH 5 hydrogels with different preservatives, theadmixture pH only increased marginally to about 7.

The surface skin pH of the admixture was also measured for 8% w/wNitricil™ NVN1 Topical IPA Gel with both a buffered and unbuffered pH 4hydrogel. The buffered pH 4 hydrogel contained Carbopol® 980P NF polymerwith 0.1% w/w citric acid. Benzoic acid and sorbic acid at 0.1% w/w werealso used as preservatives in the citric acid buffered pH4 formulatedhydrogel.

Further in vitro pH admixture measurements were taken with an unbufferedpH 4 hydrogel (Carbopol® 974P) and buffered pH 4 hydrogel with 0.1% w/wcitric acid (Carbopol® 980P) at a 1:1 ratio to confirm observations madefrom experimental results in Table 8. FIG. 2 shows that at 6% w/w and 8%w/w Nitricil™ NVN1 concentrations when used with an unbuffered pH 4hydrogel, the admixture pH is around 10 and is comparable with previousresults. The use of 0.1% w/w citric acid buffered pH 4 hydrogel has theeffect of reducing the admixture pH over a range of Nitricil™ NVN1concentrations.

An in vitro skin surface pH determination investigation was alsoperformed. FIG. 3 shows that the skin, which has a pH range of between 5and 6, provides some degree of buffering capability. Application of anunbuffered pH 4 hydrogel mixed with the 8% w/w Nitricil™ NVN1 IPATopical Gel resulted in a skin pH just below 9. The pH remainedconsistent over 30 minutes. However, when the pH 4 hydrogel bufferedwith 0.1% w/w citric acid was used (including 0.1% w/w benzoic acid and0.1% w/w sorbic acid as preservatives) with the 8% w/w Nitricil™ NVN1IPA Topical Gel, the skin surface pH was around 7.5.

Without wishing to be bound to any particular theory, the experimentsdemonstrate that with pH 4 buffered and unbuffered hydrogels at a 1:1and 3:1 ratio, the final admixture pH can be maintained between 5 to 8for Nitricil™ NVN1 concentrations ranging between 0.2% w/w and 4% w/w.With a pH 4 unbuffered hydrogel at a 3:1 ratio it is possible tomaintain the admixture pH below 8 units for Nitricil™ NVN1concentrations up to 8% w/w. With a buffered pH 6 hydrogel it ispossible to maintain the pH below 8 at Nitricil™ NVN1 concentrations of2% w/w or less. To maintain the admixture pH value below 8, aconcentration of 1% w/w Nitricil™ NVN1 or less may be used with anunbuffered pH 6 hydrogel. The use of preservatives in hydrogels has noto little effect on the admixture pH. The use of pH 4 hydrogels that areunbuffered and buffered at a ratio of 1:1 resulted in pH values greaterthan 8 units. However, when applied to the skin the pH measurement atthe surface decreases as the skin offers some buffering capacity. Toobtain a skin surface admixture pH less than 8 units, a pH 4 hydrogelthat is buffered with 0.1% w/w citric acid and containing 0.1% w/wbenzoic acid and sorbic acid can be used. However, an unbuffered pH 4hydrogel gave a skin surface pH value of greater than 8.5 when comparedto pH of 10 units from in vitro testing results.

Example 3

In vitro release testing was performed using both a single channel andmulti-channel Nitric Oxide Analyzer. An analytical balance was used toweight Nitricil™ NVN1 Topical Gel and hydrogel samples. Approximately50-mg of the Nitricil™ NVN1 Topical Gel sample and either ˜50 mg or ˜150mg hydrogel sample were transferred to a single, pre-cut weigh boatwithout allowing contact between the samples. The two samples were mixedfor approximately 5 sec., and then immediately placed into a clean, dry50-mL NO measurement cell maintained at 37° C. The real-time in vitrorelease of nitric oxide from the combined Nitricil™ NVN1 TopicalGel/Hydrogel samples was determined using the following instrumentalparameters:

-   -   1. Moist Nitrogen Flow Rate: 112-115 ml/min    -   2. Sample Temperature: 37° C.    -   3. Detection: Nitric Oxide by Chemiluminescence    -   4. Data Acquisition Frequency: 1 Hz, Irregular Sequential        Alternating    -   5. Duration: Time at which NO release rate decreases linearly        (NLT 8 hr)    -   6. Acquisition Software: NovanWare v 1.05        Conversion from parts per billion (PPB) NO to moles nitric oxide        was achieved by measuring the nitric oxide generated from a        known amount of sodium nitrite in a solution of potassium iodide        to acquire a PPB-to-mole conversion factor. Any gaps in        real-time nitric oxide release data resulting from multichannel        operation were filled in by using a linear interpolation        program. For any sample that was not measured to exhaustion of        nitric oxide, a linear extrapolation to zero release of the last        ˜5000 sec of release was performed. Real-time nitric oxide        release data was then integrated, resulting in a total nitric        oxide accumulation curve. Nitric oxide release parameters such        as C_(max) (i.e., the maximum concentration of NO released),        T_(max) (i.e., the time at which C_(max) is achieved),        Cumulative Nitric Oxide Released (i.e., the sum of all data        points per unit time), and Time to Half of Total Released (T₅₀)        (i.e., the time at which 50% of the cumulative NO is released)        were calculated from both the real time and total accumulation        nitric oxide release curves. All of the above calculations were        performed automatically in custom-built data processing software        (NovanWare v 1.05).

The results from in vitro release testing, along with the respective pHof the admixtures are summarized in Table 9 below.

TABLE 9 Results summary for pH and in vitro release testing ofNitricil ™ NVN1 topical gel. C_(max) Cumulative T_(max) T₅₀ Sample Ratio(nmol/mg/s) NO (nmol/mg) (min) (min) pH 0.2% Gel/Hydrogel pH 4 1:1 0.0095 1 14 5.5 0.2% Gel/Hydrogel pH 4 1:3 0.008 4 1 13 5.0 0.2% Gel/HydrogelpH 6 1:1 0.003 4 1 52 6.6 0.2% Gel/Hydrogel pH 6 1:3 0.007 6 1 37 6.70.5% Gel/Hydrogel pH 4 1:1 0.016 17 2 23 5.6 0.5% Gel/Hydrogel pH 4 1:30.037 17 1 7 5.1 0.5% Gel/Hydrogel pH 6 1:1 0.011 12 2 30 7.3 0.5%Gel/Hydrogel pH 6 1:3 0.014 15 1 41 7.0 1% Gel/Hydrogel pH 4 1:1 0.04237 2 17 5.8 1% Gel/Hydrogel pH 4 1:3 0.075 39 1 8 5.3 1% Gel/Hydrogel pH6 1:1 0.018 30 3 43 8.3 1% Gel/Hydrogel pH 6 1:3 0.038 41 2 51 7.2 2%Gel/Hydrogel pH 4 1:1 0.092 73 1 24 6.8 2% Gel/Hydrogel pH 4 1:3 0.06250 1 24 5.5 2% Gel/Hydrogel pH 6 1:1 0.045 29 1 50 8.6 2% Gel/HydrogelpH 6 1:3 0.070 71 2 58 8.8 4% Gel/Hydrogel pH 4 1:1 0.118 126 1 36 8.24% Gel/Hydrogel pH 4 1:3 0.175 138 2 18 7.1 4% Gel/Hydrogel pH 6 1:10.018 88 3 230 10.8 4% Gel/Hydrogel pH 6 1:3 0.068 107 2 71 9.9 6%Gel/Hydrogel pH 4 1:1 0.151 138 2 23 7.6 6% Gel/Hydrogel pH 4 1:3 0.240220 1 25 6.6 6%/Hydrogel pH 6 1:1 0.020 109 3 155 11.3 6%/Hydrogel pH 61:3 0.077 129 2 43 10.5 8% Gel/Hydrogel pH 4 1:1 0.120 180 1 67 8.3 8%Gel/Hydrogel pH 4 1:3 0.187 239 1 25 6.9 8%/Hydrogel pH 6 1:1 0.037 1345 247 10.4 8%/Hydrogel pH 6 1:3 0.056 109 9 43 10.1 12% Gel/Hydrogel pH4 1:1 0.159 242 3 94 9.6 12% Gel/Hydrogel pH 4 1:3 0.357 364 5 28 8.412% Gel/Hydrogel pH 6 1:1 0.016 184 5 371 11.4 12% Gel/Hydrogel pH 6 1:30.074 282 5 166 11.0

FIG. 4 illustrates how C_(max) from each mixture is impacted by theratio of the mixture, the pH of the hydrogel, and the concentration ofNVN1. In general, for pH 4 hydrogels the C_(max) increased withincreasing NVN1 concentration. This effect is more pronounced withmixtures containing 1:3 Nitricil™ NVN1 IPA Topical Gel to hydrogel. FIG.5 shows the increase in cumulative nitric oxide released with increasingNitricil™NVN1 concentrations.

At all Nitricil™ NVN1 concentrations, mixtures containing pH 4 hydrogelsgenerally release more of their nitric oxide payload at higher C_(max)(FIG. 4) and with a shorter half-life than with pH 6 hydrogels. Thiseffect is more pronounced for mixtures containing a 1:3 ratio with a pH4 hydrogel versus 1:1 and 1:3 ratios with a pH 6 hydrogel. FIG. 6 showsthe effects of unbuffered hydrogel pH and ratio on admixture pH withrespect to Nitricil™ NVN1 Topical Gel strength. Further in vitro studieswere performed using a pH 4 buffered citric acid hydrogel withpreservative. Measurements were repeated in triplicate. The admixturewas mixed for a period of 15 seconds prior to loading into themeasurement cell. The results for the in vitro nitric oxide releasetests are shown in Table 10.

TABLE 10 Results summary for pH and in vitro release testing ofNitricil ™ NVN1 topical gel using citric acid pH 4 buffered hydrogel.C_(max) Cumulative T_(max) T₅₀ pH of Sample (nmol/mg · s) NO (nmol/mg)(min) (min) Mixture 2% Gel/Hydrogel pH 4 (citric acid buffer) #1 0.16271 1 8 6.2 2% Gel/Hydrogel pH 4 (citric acid buffer) #2 0.109 59 1 10 2%Gel/Hydrogel pH 4 (citric acid buffer) #3 0.148 60 1 8 Average 0.140 631 9 8% Gel/Hydrogel pH 4 (citric acid buffer) #1 0.200 167 1 30 9.5 8%Gel/Hydrogel pH 4 (citric acid buffer) #2 0.218 182 1 26 8% Gel/HydrogelpH 4 (citric acid buffer) #3 0.250 185 1 23 Average 0.256 178 1 26

Example 4

A phase 1, multiple dose, single-center, observer-blind, randomized,parallel group, safety and cutaneous tolerability study was conducted in60 healthy volunteers. The objectives of this study were to evaluate thesafety and cutaneous tolerability of multiple concentrations ofNitricil™ NVN1.

The diagnosis and main criteria for inclusion into the study werehealthy male and female volunteers 18 years of age and older withelevated P. acnes counts on the face as demonstrated by a high degree offluorescence of the facial skin under a Wood's lamp.

Subjects, who had satisfied the entry criteria at the Screening/Baselinevisit, were randomized to 2% Nitricil™ NVN1 Topical Gel, 4% Nitricil™NVN1 Topical Gel, 8% Nitricil™ NVN1 Topical Gel or Topical Gel Vehiclein a 1:1:1:1 ratio (Table 11). Subjects returned once daily on theweekdays to apply the assigned treatment under supervision.Approximately 0.5 g was applied evenly over the entire face (3-4% totalbody surface area (TBSA)) after washing, sparing the eyes and mouth. OnSaturday and Sunday, subjects applied the treatment (unsupervised) athome once daily. The dose of approximately 0.5 g was topically appliedonce daily for four weeks.

TABLE 11 Test article (2% Nitricil ™ NVN1 Topical Gel, 4% Nitricil ™NVN1 Topical Gel, and 8% Nitricil ™ NVN1 Topical Gel) and reference(Topical Gel Vehicle) formulations. 2% 4% 8% Nitricil ™ Nitricil ™Nitricil ™ Topical NVN1 NVN1 NVN1 Gel Topical Gel Topical Gel TopicalGel Vehicle Ingredient (% w/w) (% w/w)¹ (% w/w) (% w/w) Isopropyl 83.5082.0x 78.50 85.5 alcohol Hexylene glycol 10.00 10.0x 10.00 10.00Cyclomethicone 2.50 2.50 2.50 2.50 Hydroxylpropyl 2.00 1.50 1.00 2.00cellulose Nitricil ™ 2.00 4.00 8.00 0 NVN1 ¹“x” denotes anon-significant FIGURE.

Assessments included cutaneous tolerability, blood chemistry andhematology, methemoglobin analysis, physical exams, urine pregnancytests (UPTs), adverse event collection and collection of vital signsincluding blood pressure. Propionobacterium acnes (P. acnes) culturesfrom the central forehead were collected at Baseline, Week 2 and Week 4.Volunteers returned for post-baseline evaluation at Weeks 2 and 4/EarlyTermination (ET).

Tolerability and safety assessments included cutaneous tolerabilityevaluation, adverse events (AEs), methemoglobin measurements, physicalexamination including vital signs, and laboratory examination [bloodchemistry, hematology and urine pregnancy tests (UPTs)].

P. acnes cultures were collected from the central forehead via a swabtechnique at Baseline (BL) Week 2, and Week 4 (Williamson 1965).

This was a preliminary study and was not powered for statisticalsignificance. All statistical processing was to be performed using SAS®unless otherwise stated. Statistical significance was based ontwo-tailed tests of the null hypothesis resulting in p-values of 0.05 orless. Safety analyses were performed using the safety population.

Each Cutaneous Tolerability Scale was summarized categorically andcontinuously, by treatment group and visit. Within the categoricalsummary, a Cochran-Mantel-Haenszel test, using modified ridits, wasperformed for each post-baseline assessment.

Blood chemistry and hematology values were reported individually atBaseline and at Weeks 2 and 4/ET. Additionally, the change from baselinewas reported at Week 2 and Week 4/ET. Labs were reported in SI units.

Methemoglobin was summarized categorically and continuously, bytreatment group and visit. Additionally, box-plots were presented bytreatment group and visit. The boxplots displayed the mean, median,minimum, maximum, first and third quartiles, with all observed datapoints overlayed upon the boxplot so that any outliers were easilyvisualized.

All AEs occurring during the study were recorded and classified on thebasis of the Medical Dictionary for Regulatory Authorities (MedDRA)terminology. All reported AEs that occurred on or after the Baselinedate through the end of study visit were included in the summaries andanalysis. If an event occurred prior to the administration of studydrug, it was considered medical history and was listed and summarized assuch.

Adverse event summaries included the number and percentage of subjectswho reported at least one AE and the number of events reported byseverity, seriousness, and relationship to study medication. The summaryof adverse events was also presented by system organ class (SOC) andpreferred terms (PT) based on MEDDRA version 15.1. A subject was countedonly once under each SOC and PT.

Adverse event SOCs and PTs were summarized by severity and relationshipto investigational product. For the summary by severity, subjects wereonly counted once for a specific SOC or PT under the worst severity.Severity was collected as mild, moderate, severe, or life-threatening.For the summary by relationship to investigational product, subjectswere only counted once for a specific SOC or PT under the highestrelationship. Relationship was collected as definite, probable,possible, unlikely, unrelated, or not applicable. For the summary ofrelationship, definite, probable, and possible were considered relatedand unlikely, unrelated, and not applicable was considered unrelated.

All information pertaining to AEs noted during the study were listed bysubject, detailing verbatim given by the Investigator, preferred term,system organ class, start date, stop date, severity, seriousness, andrelationship to investigational product. The AE onset was also shownrelative (in number of days) to the day of initial dose of therandomized investigational product. A summary of adverse events thatlead to a subject's discontinuation of investigational product usage wasalso provided.

P. acnes Counts:

Quantitative bacteriologic cultures were obtained from the forehead atBaseline and at Weeks 2 and 4/ET. Samples were obtained according to amodification of the technique of Williamson and Kligman and cultured for7 days (Williamson 1965). Colony forming units (cfu) of P. acnes werecounted at the dilution that contained between 10 and 100 cfu. Totaldensities of P. acnes were calculated and reported as log 10 cfu percm².

Descriptive statistics of P. acnes, change from baseline, and percentchange from baseline were summarized with mean, median, standarddeviation, minimum, maximum and 95% confidence intervals. The per centreduction from baseline at Week 2 and Week 4 were calculated using thefollowing formula:

$\frac{\left( {1 \times 10^{X}} \right) - \left( {1 \times 10^{Y}} \right)}{\left( {1 \times 10^{X}} \right)}$

Where X=the initial Log value, and Y=the final Log value.

Safety & Tolerability:

In this study, Nitricil™ NVN1 Topical Gel was demonstrated to be safeand well-tolerated. The majority of subjects in the Nitricil™ NVN1Topical Gel and Vehicle Gel treatment groups did not experienceerythema, scaling, dryness, pruritus, or burning/stinging during thetreatment period. Scores other than none were generally mild with 1subject reporting moderate erythema.

A total of 17 AEs were reported by 12 subjects in the Nitricil™ NVN1Topical Gel treatment groups and 4 AEs were reported by 4 subjects (27%)in the Vehicle Gel treatment group. The most frequent reported AEs werenasal congestion and headache. All AEs were mild or moderate inseverity, were judged by the investigator to be unrelated to studymedication, required no action to be taken with respect to studymedication, and were resolved at the end of the study. No AEs wereserious.

For the Nitricil™ NVN1 Topical Gel treatment groups, the methemoglobinlevel averaged 0.77-0.91% at Baseline, 0.73-0.97% at Week 2, and0.67-0.85% at Week 4. For the Vehicle Gel treatment group, themethernoglobin level averaged 0.77, 0.91, and 0.83% at Baseline, Week 2,and Week 4, respectively. The highest methemoglobin observed (1.9%)during the study was in a subject treated with Vehicle Gel.

P. acnes Counts:

There was no difference in P. acnes counts in Nitricil™ NVN1 treatedsubjects versus Topical Gel Vehicle treated groups. The averagereduction in P. acnes between Baseline and Week 4 was: 57% (GelVehicle); 58% (2% Nitricil™ NVN1); 54% (4% Nitricil™ NVN1); and 63% (8%Nitricil™ NVN1).

Nitricil™ NVN1 was safe and well-tolerated in this study. There were nodifferences found between treatment groups in tolerability, reportedadverse events, laboratory results including methemoglobinconcentrations, or physical examinations including vital signs. Therewas no difference in percent reduction in P. acnes counts from baselineat Week 2 and Week 4 between Nitricil™ NVN1 Topical Gel treated groupsand Topical Gel Vehicle.

Example 5

This study was a phase 1, single-center, evaluator-blind, randomized,parallel group, safety and cutaneous tolerability study conducted in 30healthy volunteers with elevated Propionobacterium acnes counts. Theobjectives of the study were to evaluate the safety and cutaneoustolerability of Nitricil™ NVN1 Gel.

Subjects who satisfied the entry criteria at the Screening and Baselinevisits were randomized to 4% Nitricil™ NVN1 Gel or Vehicle Gel in a 2:1ratio (Table 12). Subjects returned once daily to apply the assignedtreatment under supervision. Approximately 1 g was applied over theentire face (3-4% total body surface area (TBSA)), sparing the eyes andmouth, twice daily.

TABLE 12 Test article (4% Nitricil ™ NVN1 Gel) and reference (VehicleGel) formulations in a dual chamber pump. Nitricil ™ NVN1 4% Gel VehicleGel Ingredient (% w/w) (% w/w) Chamber A Isopropyl alcohol 39.25 42.725Hexylene glycol 5.00 5.00x¹ Cyclomethicone 1.25 1.25x Hydroxylpropylcellulose 0.50 1.00x Nitricil ™ NVN1 4.00 0 Titanium dioxide (Opacifier)0 0.025 Chamber B Purified Water 42.50 42.50x Glycerin 5.00 5.00xCarbomer Homopolymer 0.50 0.50x Type C Carbopol ® 980 Citric Acid,anhydrous 0.05 0.05x Benzoic Acid 0.05 0.05x Sorbic Acid 0.05 0.05xTrolamine QS¹ QS² Purified Water QS² QS³ Total 100.0 100.0 ¹“x” denotesa non-significant digit. ²Quantity sufficient to pH 4 for Chamber B.³Quantity sufficient to make total.

Assessments included cutaneous tolerability, methemoglobin andhemoglobin analysis, physical exams including vital signs, and adverseevent collection.

P. acnes cultures from the central forehead were collected at Baselineand post-treatment (Weeks 1 and 2). Subjects returned for post-Baselineevaluation at Week 1 and Week 2/Early Termination (ET).

The diagnosis and main criteria for inclusion were healthy male andfemale volunteers 18 years of age and older with elevated P. acnescounts on the face as demonstrated by a high degree of fluorescence ofthe facial skin under a Wood's lamp

Tolerability and safety assessments included cutaneous tolerabilityevaluation, adverse events (AEs), hemoglobin and methemoglobinmeasurements, and physical examination including vital signs.

P. acnes cultures were collected from the central forehead via a swabtechnique at Baseline (BL), Week 1 and Week 2/ET (Williamson 1965).

This was a preliminary study and was not powered for statisticalsignificance. However, the sample size is sufficiently large enough todetect an average 0.75 difference in scores between treatment groups inany of the cutaneous tolerability assessments with 80% power atalpha=0.05. All statistical processing was performed using SAS® unlessotherwise stated. Statistical significance was based on two-tailed testsof the null hypothesis resulting in p-values of 0.05 or less, unlessotherwise specified. Safety analyses was performed using the safetypopulation.

Cutaneous tolerability assessments (erythema, scaling, dryness,pruritus, burning/stinging) were summarized with frequency counts andpercentages at each evaluation for each score category at Week 1 andWeek 2. Comparison of treatment differences in distribution of scoreswas performed using Cochran-Mantel-Haenszel (CMH) chi-square test withMODRIDIT option for ordered scores.

Total hemoglobin was reported; methemoglobin was reported as apercentage of hemoglobin. Hemoglobin and methemoglobin were summarizeddescriptively by treatment group at Screening and Weeks 1 and 2 andinclude n, mean, median, standard deviation, minimum, and maximum.Additionally, the change from baseline in hemoglobin and methemoglobinat Weeks 1 and 2 were summarized. Comparison of treatment differences inchanges from baseline hemoglobin and methemoglobin were performed usingWilcoxon Rank Sum test.

Quantitative bacteriologic cultures were obtained from the forehead atBaseline and at Weeks 1 and 2. Samples were obtained according to amodification of the technique of Williamson and Kligman and cultured for7 days (Williamson, B. A.; Kligman, A. M. A new method for thequantitative investigation of cutaneous bacteria. J. Invest. Dermatol.1965, 45, 498-503). Colony forming units (cfu) of P. acnes were countedat the dilution that contained between 10 and 100 cfu. Total densitiesof P. acnes were calculated and reported as log₁₀ cfu per cm².

Descriptive statistics of P. acnes, change from Baseline, and percentchange from Baseline were summarized with mean, median, standarddeviation, minimum, maximum and 95% confidence intervals.

The per cent reduction from Baseline at Week 1 and Week 2 werecalculated using the following formula:

$\frac{\left( {1 \times 10^{X}} \right) - \left( {1 \times 10^{Y}} \right)}{\left( {1 \times 10^{X}} \right)}$

Where X=the initial Log value, and Y=the final Log value.

In this study, Nitricil™ NVN1 4% Gel was demonstrated to be safe andwell-tolerated. The majority of subjects in Nitricil™ NVN1 4% Gel andVehicle Gel treatment groups did not experience erythema, scaling,dryness, pruritus, or burning/stinging during the treatment period.

No adverse events were reported in this study. No clinically significantchange in blood pressure, pulse or findings on physical exam were seenbetween Baseline and the end of treatment. No clinically significantchanges in percent methemoglobin or hemoglobin concentration were found.

After 1 week of treatment, there was a mean logarithm reduction in P.acnes counts of 0.38 for the Nitricil™ NVN1 4% Gel group compared to0.20 for the Vehicle Gel group. After 2 weeks, subjects treated withNitricil™ NVN1 4% Gel had a mean reduction in P. acnes counts of 0.51log₁₀ cfu per cm² compared to 0.26 log₁₀ cfu per cm² for the subjectstreated with Vehicle Gel. The difference at Week 2 was statisticallysignificant, p=0.04, using a Student's T-Test. A post-hoc ANCOVA alsodemonstrated a statistically significant difference in P. acnesreduction in subjects treated with Nitricil™ NVN1 4% Gel and VehicleGel, p=0.03.

Nitricil™ NVN1 4% Gel was safe and well-tolerated in this study. Therewas a statistically significant difference between Nitricil™ NVN1 4% Geltreated groups and Vehicle Gel treated groups in the percent reductionin P. acnes counts.

Example 6

The primary objective of this study was to evaluate the cutaneoustolerability of Nitricil™ NVN1 4% Gel (containing a hydrogel) atBaseline and Weeks 1 and 2/ET. The secondary objective was to evaluatethe safety profile of Nitricil™ NVN1 Gel. Safety was assessed bycomparing adverse events between groups (including clinicallysignificant changes in physical exams and vital signs), changes inhemoglobin and the percent methemoglobin. Exploratory analysis ofefficacy as measured by reduction of P. acnes as determined by changesin the number of organisms from cultures at the Baseline Visit, Week 1and Week 2/ET was performed.

The study panel had 30 subjects who were healthy adult males and females18 years of age and older and who were colonized by P. acnes. Subjectswere carefully screened to ensure that none were using any prohibitedtopical or systemic antibiotics within 4 weeks prior to enrollment. Thepanelists were instructed not to use any medicated shampoos. Thevolunteers selected for the study showed a high degree of fluorescenceof the facial skin under a Wood's lamp indicating the presence of highlevels of P. acnes. Baseline P. acnes counts were at least 10,000colonies per cm² on the facial skin.

All subjects met the below inclusion/exclusion criteria.

-   Inclusion Criteria:    -   Have a signed written informed consent form.    -   Be a healthy, adult male or female volunteer, 18 years of age        and older.    -   Show a high degree of fluorescence of the facial skin under a        Wood's lamp indicating the presence of high levels of P. acnes.    -   Have no past or present history of any significant internal        disease (e.g. cardiovascular, pulmonary, renal, etc.).    -   If a woman of childbearing potential (WOCBP), have a negative        urine pregnancy test (UPT) at Baseline.    -   If a WOCBP, agree to use an effective method of birth control        during the course of the study and for 30 days after the final        study visit. Females taking hormonal contraceptives must have        taken the same type for at least three months (90 days) prior to        entering the study and must not change type during the study.        Those who have used hormonal contraceptives in the past and        stopped must have discontinued usage at least three months prior        to the start of the study.    -   Agree to refrain from using antimicrobial topical products        (shampoos, soaps, acne preparations, etc.).    -   Be compliant and able to return to site as instructed once daily        (Monday-Friday) for approximately two weeks.-   Exclusion Criteria:-   Subjects were not enrolled if they met any of the following    exclusion criteria:    -   Exhibit any skin disorders of an acute or chronic nature        including psoriasis, eczema, etc.    -   Have a history of experiencing significant burning or stinging        when applying any facial treatment (e.g., make-up, soap, masks,        washes, sunscreens, etc.) to their face.    -   Female subjects who are pregnant, nursing mothers, or planning        to become pregnant during the study.    -   Male subjects who do not agree to sexual abstinence, refraining        from sperm donation and/or using a barrier (male condom)        throughout the study.    -   Have used estrogens (e.g., Depogen, Depo-Testadiol, Gynogen,        Valergen, etc.) or oral contraceptives for less than 90 days        immediately preceding the Baseline visit, discontinued use of        estrogens or oral contraceptives less than 90 days prior to        Baseline, or planning to begin or discontinue use of this        therapy during the treatment period.    -   Have used topical or systemic antibiotics within the previous 4        weeks that are known to influence P. acnes counts e.g.        minocycline, tetracycline, erythromycin, clindamycin,        doxycycline etc.    -   Use of other medications which may influence skin surface P.        acnes levels (e.g., retinoids) within the previous 6 months.    -   Use nitroglycerin or other nitric oxide donor drug        concomitantly.    -   Have a clinically significant anemia (as determined by the        principal investigator) at Baseline.    -   Have a Screening methemoglobin value of ≧2.0%.    -   Have clinically significant anemia at Screening as determined by        the Investigator.    -   Are known to be allergic to any of the components in the        investigational product.    -   Have intercurrent illness requiring administration of prohibited        antibiotics.    -   Have any condition or situation which, in the Investigator's        opinion, puts the subject at significant risk, could confound        the study results, or may interfere significantly with the        subject's participation in the study. Subjects undergoing        endoscopy with use of topical anesthetics should not be enrolled        in and should be discontinued from the study prior to endoscopy.        Subjects with clinically significant anemia, as determined by        the Investigator, should not be enrolled.    -   Are unable to communicate or cooperate with the Investigator due        to language problems, poor mental development, or impaired        cerebral function.    -   Have used an investigational drug or device within 30 days of        enrollment or concurrent participation in a different research        study.

Volunteers were free to withdraw their consent and discontinueparticipation in the study at any time.

Candidates were screened for eligibility prior to enrollment. Afterensuring qualification and signing an informed consent, quantitativeBaseline measurements of P. acnes (on the forehead) were obtained (seesection below “Quantitative Bacteriology”).

Treatment Plan:

Treatment was for two weeks_(—) Each weekday morning under supervisionby a technician at the Skin Study Center, the volunteer washed theirface and then applied the test product. Approximately 1 g of Nitricil™NVN1 4% Gel or Gel Vehicle (Table 12) was applied evenly over the entireface (4% TBSA), sparing the eyes and mouth. The investigational productwas dispensed from a dual chamber pump by depressing the pump 3 times.The dispensed material was quickly (3-5 seconds) mixed together andapplied in a thin layer to the face, sparing the eyes and mouth. Theproduct was then gently massaged into the skin for about 30 seconds.Subjects washed their hands after study drug application. This procedureof washing the face, applying the test product and then washing of handswas done in the evening by the panelists at home (no supervision).

All treatments were documented on the Subject Diary.

Quantitative Bacteriology:

Quantitative bacteriologic cultures were obtained from the test sites atBaseline (0) and at Weeks 1 and 2. Samples were obtained according to amodification of the technique of Williamson and Kligman {Williamson, P.and Kligman, A. M.: A new method for the quantitative investigation ofcutaneous bacteria. Journal of Investigative Dermatology 45: 498-503,1965; Keyworth N., Millar, M. R., and Holland, K. T.: Swab-Wash Methodfor Quantitation of Cutaneous Microflora. J. Clin. Microbiology, Vol.28, pp. 941-943, 1990). The forehead was cleansed of surface bacteria bythoroughly wiping the area for 30 seconds with sterile gauze soaked with0.1% Triton-X-100 to remove surface debris and bacteria. The surfacearea to be cultured (4 cm2) was then delineated by a sterile plastictemplate held firmly to the skin. A sterile cotton-tipped swab wasdipped into 2 ml of wash solution (Bacto Letheen Broth, Difco, Sparks,Md., USA). The area was then scrubbed with the cotton-tipped swab for 30seconds. The swab was then placed back into the 2 ml wash solution andwrung on the side of the tube. The same skin area was then scrubbedagain for another 30 seconds after which the cotton-tipped swab is againplaced back into the 2 ml of wash solution and wrung on the side of thetube. The swab was then broken off into the 2 ml of wash solution. Thesample was subsequently processed as described in the Williamson-Kligmanmethod viz. the wash sample was serially diluted using 0.05% Tween-80(buffered with 0.075M phosphate buffer, pH 7.9) in 4 ten-fold dilutions.Using a micropipettor, 0.05 mL of each dilution was placed on adesignated section of an agar plate containing Brucella agarsupplemented with yeast extract, dextrose, and cysteine, five dropdilutions per plate. Duplicate plates were made for each subject. Plateswere allowed to dry, placed in an anaerobic jar with BBL Gas Pak Plusanaerobic system envelope and incubated anaerobically at 36.5-37.50 Cfor 7 days. Colony forming units (cfu) of P. acnes are counted at thedilution that contains between 10 and 100 cfu. Total densities of P.acnes were calculated and reported as log₁₀ cfu per cm².

Cutaneous Tolerability Assessment:

At Baseline, Week 1 and Week 2, cutaneous tolerability assessments weremade by the dermatologist prior to treatment. Cutaneous tolerabilityendpoints were not reported as an AE unless they reached severe and/orresult in subject's discontinuation from the study. Cutaneoustolerability assessments were performed according to the followingscales:

Erythema

-   0-None No evidence of erythema present-   1-Mild Slight pink coloration-   2-Moderate Definite redness-   3-Severe Marked erythema, bright red to dusky dark red in color

Scaling

-   0-None No scaling-   1-Mild Fine scales present to limited areas of the face, barely    perceptible-   2-Moderate Fine scale generalized to all areas of the face-   3-Severe Scaling and peeling of skin over all areas of the face

Dryness

-   0-None No dryness-   1-Mild Slight but definite roughness-   2-Moderate Moderate roughness-   3-Severe Marked roughness

Pruritus

-   0-None No itching-   1-Mild Slight itching, not very bothersome-   2-Moderate Moderate amount of itching, somewhat bothersome-   3-Severe Severe amount of itching, definite discomfort and sleep may    be disturbed

Burning/Stinging

-   0-None No burning/stinging-   1-Mild Slight warm, burning/stinging sensation; not very bothersome-   2-Moderate Definite warm, burning/stinging sensation that is    somewhat bothersome-   3-Severe Hot, tingling/sensation that has caused definite discomfort    and may have disturbed sleep

Laboratory Assessments:

Hemoglobin was measured at Baseline and at Weeks 1 and 2/ET using aMasimo Rainbow® SET® Rad57™ pulse co-oximeter. The amount of hemoglobin(g/dL) was displayed on the pulse co-oximeter and recorded on the CRF.

Methemoglobin was measured at Baseline and at Weeks 1 and 2/ET using aMasimo Rainbow® SET® Rad-57™ pulse co-oximeter. The percentmethemoglobin was displayed on the pulse co-oximeter and recorded on theCRF.

All WOCBP had a urine pregnancy test (UPT) at Baseline and at theirfinal evaluation visit (Week 2/ET).

A brief physical exam was performed at Baseline and Week 2/ET.

Systolic and diastolic blood pressure and pulse were collected atBaseline and Weeks 1 and 2/ET.

Data Processing:

Cultures were plated in duplicate with tenfold dilutions from 100 to104. Bacterial counts were obtained from the dilution at which colonyforming units (CFUs) are significantly dispersed and then converted tototal number per sq. cm. The raw data for each plate and the average wasprovided in separate columns. Sorting templates were based on Excel 2007spreadsheet software. The sorted data for each session were tabulated.

Data analysis involved a Paired T-Test to comparing Baseline means withtreatment points. A Student's T-Test was used to compare means for thetwo treatments. A two tailed p<0.05 is required for a significantdifference.

Thirty-three (33) panelists were screened for this study and a total ofthirty (30) panelists were enrolled. Twenty-nine (29) panelistscompleted. One panelist withdrew consent from the study due to rednessafter application. There were no adverse events.

No erythema or scaling was observed by the dermatologist during thecourse of this study. Minimal dryness, pruritus and burning or stingingwas noted as follows.

Three subjects using Nitricil™ NVN1 4% Gel (Treatment A) reporteddryness at Week 2. Two subjects using Treatment A reported pruritus atWeek 1. At week 1, one subject reported burning/stinging using TreatmentA.

In this trial, the cutaneous tolerability and systemic safety of a 4%Nitricil™ NVN1 gel formulation was evaluated after 1 and 2 weeks ofB.I.D. treatment. In addition, an exploratory analysis of in-vivoreduction of Propionibacterium acnes, the organism responsibility forthe inflammation in acne vulgaris, was done after 1 and 2 weeks oftreatment. The test panel consisted of 30 healthy volunteers with P.acnes levels of 10⁴/cm² or greater; 20 were treated with the activeagent and 10 with the vehicle.

The test agent and its vehicle were extremely well tolerated. Noobjective signs of irritation and no subjective symptoms of pruritus,burning or stinging were seen except for moderate pruritus in 1 panelistand mild in another panelist at the Week 1 visit. Mild burning/stingingwas reported by one panelist at Week 1. Mild dryness was seen in 3panelists at Week 2. One panelist withdrew on Day 1 because of concernsabout the physiological vasodilatation induced by the test agent. Nosign of irritation was seen in this panelist.

No clinically significant change in blood pressure, pulse or findings onphysical exam were seen between Baseline and the end of treatment. Noclinically significant changes in percent methemoglobin or hemoglobinconcentration were found.

After 1 week of treatment, there was a mean logarithm reduction of 0.38for the active agent compared to 0.20 for the vehicle. After 2 weeks,the active agent had a mean reduction of 0.51 compared to 0.26 for thevehicle. The difference at Week 2 was statistically significant using astudent's T-test (p=0.04). The response was variable in test agent with2 panelists showing greater than 1 log reduction after 2 weeks oftreatment, 5 others with 0.5 or greater but less than 1 log reductionand 3 panelists showing less than the mean reduction in the vehiclegroup.

An analysis of co-variance (ANCOVA) demonstrated statisticallysignificant differences in for P. acnes counts at week 1 and week 2(FIG. 9).

The test agent and its vehicle were well tolerated and showed no signsof cutaneous or systemic toxicity. A variable in-vivo antibacterialeffect in P. acnes was seen which may be of benefit in acne therapy.

Example 7

This study was a multi-center, randomized, evaluator-blinded,vehicle-controlled, parallel group, three-arm study to compare theefficacy, safety, and tolerability of two concentrations of Nitricil™NVN1 Gel and Vehicle Gel in subjects with acne vulgaris treated for 12weeks. Subjects who met the study entry criteria were enrolled andrandomized to receive topical applications of Nitricil™ NVN1 1% Gel,Nitricil™ NVN1 4% Gel, or Vehicle Gel (Table 13). Subjects wererandomized in a 1:1:1 ratio to Nitricil™ NVN1 1% Gel, Nitricil™ NVN1 4%Gel, or Vehicle Gel and instructed to dose twice daily (morning andnight) for 12 weeks (84 days). The first application of study drug tookplace at the investigational site at the Baseline visit.

TABLE 13 Test article (Nitricil ™ NVN1 1% Gel and Nitricil ™ NVN1 4%Gel) and reference (Vehicle Gel) formulations in a dual chamber pump.Nitricil ™ Nitricil ™ NVN1 1% NVN1 4% Gel Gel Vehicle Gel Ingredient (%w/w) (% w/w) (% w/w) Chamber A Isopropyl alcohol 41.75 39.25 42.725Hexylene glycol 5.00 5.00 5.00x¹ Cyclomethicone 1.25 1.25 1.25xHydroxylpropyl cellulose 1.00 0.50 1.00x Nitricil ™ NVN1 1.00 4.00 0Titanium dioxide (Opacifier) 0 0 0.025 Chamber B Purified Water 42.5042.50 42.50x Glycerin 5.00 5.00 5.00x Carbomer Homopolymer Type C 0.500.50 0.50x Carbopol ® 980 Citric Acid, anhydrous 0.05 0.05 0.05x BenzoicAcid 0.05 0.05 0.05x Sorbic Acid 0.05 0.05 0.05x Trolamine QS¹ QS¹ QS²Purified Water QS² QS² QS³ Total 100.0 100.0 100.0 ¹“x” denotes anon-significant digit. ²Quantity sufficient to pH 4 for Chamber B.³Quantity sufficient to make total.

After the Baseline visit, study visits took place approximately everytwo weeks for the first four weeks, then every four weeks for the nexteight weeks. The study duration was up to 84 days of treatment.

Efficacy assessments included inflammatory (papules and pustules) andnoninflammatory (open and closed comedones) lesion counts, nodules andcysts counts, and Investigator's Global Assessment (IGA), which wereperformed at Baseline and Weeks 4, 8, and 12/Early Termination (ET).

Additional assessments included photographs and sebum collection.Photographs were collected at Baseline, Week 4 and Week 12/ET. Sebum wascollected from the central forehead of subjects enrolled at twoinvestigational sites at Baseline, Week 4, and Week 12/ET usingSebutapes®.

Tolerability and safety assessments included cutaneous tolerabilityevaluation, adverse event (AE) collection, physical exams includingblood pressure and pulse rate, methemoglobin and hemoglobinmeasurements, and urine pregnancy tests (UPTs). Cutaneous tolerabilityassessments (erythema, scaling, dryness, pruritus, and burning/stinging)were evaluated prior to and 30 minutes after the first application ofstudy drug at the Baseline visit and at each subsequent visit. Thecutaneous tolerability assessments for visits other than the Baselinewere to be performed at least 30 minutes after study drug application.AEs were collected starting after the subject had signed the informedconsent and completed any study assessment until the end of the finalstudy visit. A brief physical exam was performed at Baseline (Visit1/Day 0) and Week 12/ET. Blood pressure and pulse rate were collectedpre-dose at Baseline and at Weeks 2, 4, 8, and 12/ET. Methemoglobin andhemoglobin were measured at Baseline, Week 2 and Week 12/ET using aMasimo Rainbow® SET® Rad57™ pulse co-oximeter that analyzedmethemoglobin and hemoglobin levels. Subjects with methemoglobin valuesof >2.0 percent at Baseline and subjects with clinically significantanemia at Baseline were not eligible to participate in the study. Allwomen of child-bearing potential (WOCBP) must have had a UPT at Baselineand if the result was positive, the subject was not allowed toparticipate in the study. WOCBP also were to have a UPT at Weeks 4, 8,and 12/ET. Subjects who terminated early were asked to complete all Week12/ET evaluations at the time of premature discontinuation.

The number of subjects in the intent to treat population were: 52subjects for the Vehicle Gel group, 51 subjects for the Nitricil™ NVN11% Gel group, and 50 subjects for the Nitricil™ NVN1 4% Gel group.However, not all of the subjects completed the study, and thus 45subjects completed the study for the Vehicle Gel group, 43 subjectscompleted the study for the Nitricil™ NVN1 1% Gel group, and 41 subjectscompleted the study for the Nitricil™ NVN1 4% Gel group.

The study included healthy males and females of any race who were 12 to40 years of age (inclusive), with acne vulgaris. Subjects must have hadat least 20 but no more than 40 inflammatory lesions (papules andpustules), 25 to 70 noninflammatory lesions (open and closed comedones),no more than two nodules, and investigator's global assessment (IGA) of2, 3, or 4.

The Nitricil™ NVN1 1% Gel, Nitricil™ NVN1 4% Gel, and Vehicle Gel wereadministered by applying approximately 1 gram evenly over the entireface twice a day (morning and evening). The active gel (Chamber A) andhydrogel (Chamber B) were dispensed concurrently from a dual chamberpump in a ratio of 1:1 and mixed by the subject immediately prior toapplication. The Vehicle Gel (Chamber A) and hydrogel (Chamber B) weredispensed concurrently from a dual chamber pump and mixed by the subjectimmediately prior to application.

At Baseline and Weeks 4, 8, and 12/ET, the investigator counted thetotal number of noninflammatory lesions on the subject's face includingthe forehead, right and left cheeks, chin and nose. At Baseline andWeeks 4, 8, and 12/ET, the investigator also counted the total number ofinflammatory lesions on the subject's face, including the forehead,right and left cheeks, nose, and chin. The lesion count for the nose(inflammatory and noninflammatory) and the number of nodules and cystswere reported separately, but for analyses, the inflammatory lesioncount included the collective number of papules, pustules, andnodules/cysts.

The IGA was performed at Baseline and Weeks 4, 8, and 12/ET. The IGAscore was determined based on the investigator evaluation of the overallsigns and symptoms of acne vulgaris. Evaluations were scored on a scaleof 0 (clear) to 4 (severe).

Photographs were collected at Baseline, Week 4 and Week 12/ET.

Sebum was collected from the central forehead of subjects enrolled attwo investigational sites at Baseline, Week 4, and Week 12/ET usingSebutapes®.

The investigator evaluated the subject's face prior to and 30 minutesafter the first application of investigational product in addition toevaluating at each study visit. The cutaneous tolerability assessmentfor visits other than the Baseline should have been performed at least30 minutes after study drug application. Cutaneous tolerabilityevaluations included erythema, scaling, dryness, pruritus, andburning/stinging.

Methemoglobin and hemoglobin were measured at Baseline, Week 2 and Week12 using a Masimo Rainbow® SET® Rad-57™ pulse co-oximeter that analyzedmethemoglobin and hemoglobin levels.

A brief physical exam was performed at Baseline {Visit 1/Day 0) and Week12/ET. Blood pressure and pulse rate were collected pre-dose at Baselineand at Weeks 2, 4, 8, and 12/ET. The investigator assessed subjects ateach scheduled study visit for the occurrence of AEs.

The primary efficacy endpoint was absolute change from baseline innon-inflammatory lesion count on the face including the nose at Week 12.

Analysis of the primary endpoint of absolute change in non-inflammatorylesion count on the face including the nose at Week 12 was conductedusing an analysis of covariance (ANCOVA) with factors of treatment andinvestigational site and baseline lesion count as a covariate. A linearregression also was performed to determine dose response where the slope(β) was estimated across treatments (1% and 4%) and where the VehicleGel was labeled as 0% for the regression. The null test was that (3=0versus the alternative that β is not equal 0. Rejection of the nullhypothesis with a positive β indicated a dose response.

Secondary efficacy endpoints were: Absolute change from baseline ininflammatory lesion count at Week 12, absolute change from baseline innon-inflammatory lesion count on the face excluding the nose at Week 12,and dichotomized IGA at Week 12.

Inflammatory lesion count included the collective number of papules,pustules, and nodules/cysts.

The analysis of the absolute change in inflammatory lesion counts atWeek 12 used the same methods outlined for the primary efficacy analysisof change in noninflammatory lesion counts including the nose.

The analysis of the absolute noninflammatory lesion count on the faceexcluding the nose at Week 12 used the same methods outlined for theprimary efficacy analysis of change in noninflammatory lesion counts onthe face including the nose.

The dichotomized IGA scores at Week 12 were analyzed using a logisticregression model where treatment and investigational sites were theindependent factors. The dichotomized IGA score was relabeled as 0 forfailure and 1 for success as the dependent variable in the logisticregression. Success was defined as a score of ‘clear’ (0) or ‘almostclear’ (1) with at least a two-grade improvement from baseline.Additionally, treatment groups were compared using theCochran-Mantel-Haenszel (CMH) test stratified by investigational site.Pairwise comparisons were computed without concern for controlling formultiplicity.

Cutaneous tolerability assessments (erythema, scaling, dryness,pruritus, and burning/stinging) were summarized as frequency counts andpercentages at each evaluation. Methemoglobin was reported as apercentage of hemoglobin. Total hemoglobin also was reported.Methemoglobin and hemoglobin were summarized descriptively by treatmentgroup at Baseline and Weeks 2 and 12 and included sample size, mean,median, standard deviation (SD), minimum, and maximum. Additionally, thechanges from baseline in methemoglobin and hemoglobin at Weeks 2 and 12were summarized.

Summaries were presented to describe the characteristics of the AEsreported and included the number and percentage of subjects who reportedat least one AE and the number of events reported by severity,seriousness, and relationship to study drug.

Blood pressure and pulse rate were summarized at Baseline, Week 2, Week4, Week 8, and Week 12/ET as well as the change from Baseline at Week12/ET by treatment group with mean, SD, minimum, and maximum.

For the ITT population, the Nitricil™ NVN1 1% Gel and Nitricil™ NVN1 4%Gel LSMean changes from baseline for absolute noninflammatory lesions(including and excluding the nose) were statistically significantlylarger decreases than the corresponding Vehicle Gel LSMean changes frombaseline for absolute noninflammatory lesions (including and excludingthe nose), but linear dose responses were not seen. The Nitricil™ NVN14% Gel LSMean change from baseline for absolute inflammatory lesions wasa statistically significantly larger decrease than the Vehicle GelLSMean change from baseline for absolute inflammatory lesions, but theNitricil™ NVN1 1% Gel LSMean change from baseline for absoluteinflammatory lesions was not statistically different from the VehicleGel LSMean change from baseline for absolute inflammatory lesions. Alinear dose response was seen. No differences were seen among treatmentgroups for the dichotomized IGA. Specifically,

-   -   The LSMean absolute change from baseline for noninflammatory        lesions (including nose) was −11.8 lesions for the Nitricil™        NVN1 1% Gel group compared to −0.7 lesions for the Vehicle Gel        group (p=0.022). The LSMean absolute change from baseline for        noninflammatory lesions (including nose) was −11.1 lesions for        the Nitricil™ NVN1 4% Gel group compared to −0.7 lesions for the        Vehicle Gel group (p=0.031). A linear dose response was not seen        among the treatment groups 0)-0.105). The LSMean absolute change        from baseline for inflammatory lesions was −13.8 lesions for the        Nitricil™ NVN1 1% Gel group compared to −9.4 lesions for the        Vehicle Gel group (p=0.088). The LSMean absolute change from        baseline for inflammatory lesions was −15.5 lesions for the        Nitricil™ NVN1 4% Gel group compared to −9.4 lesions for the        Vehicle Gel group (p=0.018). A linear dose response was seen        among the treatment groups (p=0.033).    -   The LSMean absolute change from baseline for noninflammatory        lesions (excluding nose) was −10.9 lesions for the Nitricil™        NVN1 1% Gel group compared to −1.3 lesions for the Vehicle Gel        group (p=0.032). The LSMean absolute change from baseline for        noninflammatory lesions (excluding nose) was −10.6 lesions for        the Nitricil™ NVN1 4% Gel group compared to −1.3 lesions for the        Vehicle Gel group (p=0.039). A linear dose response was not seen        among the treatment groups (p=0.118).    -   0.0% (0/51) subjects were characterized as success for the        dichotomized IGA for the Nitricil™ NVN1 1% Gel group compared to        19% (1/52) subjects for the Vehicle Gel group (p≧0.317). 2.0%        (1/50) subjects were characterized as success for the        dichotomized IGA for the Nitricil™ NVN1 4% Gel group compared to        1.9% (1/52) subjects for the Vehicle Gel group (p>0.601).

For the Safety population, Nitricil™ NVN1 1% Gel and Nitricil™ NVN1 4%Gel were safe and generally well tolerated. There were no seriousadverse events and the Nitricil™ NVN1 1% Gel and Nitricil™ NVN1 4% Gelshowed good cutaneous tolerability. A review of the methemoglobin andhemoglobin results did not identify any safety signals. A review of thevital signs results did not identify any safety signals.

For the PP population, Nitricil™ NVN1 1% Gel and Nitricil™ NVN1 4% Gelboth demonstrated a mean percentage reduction for noninflammatorylesions over time compared to the Vehicle Gel (FIG. 10). FIG. 10 showsthat separation from the Vehicle Gel group occurred at week 4 for boththe Nitricil™ NVN1 1% Gel and Nitricil™ NVN1 4% Gel groups. At week 4,the Vehicle Gel group had a 6% mean percentage reduction fornoninflammatory lesions, whereas the Nitricil™ NVN1 1% Gel group had a14% mean percentage reduction for noninflammatory lesions and theNitricil™ NVN1 4% Gel group had a 24% mean percentage reduction fornoninflammatory lesions. The noninflammatory lesion count for the PPpopulation is provided in Table 14.

TABLE 14 Summary of noninflammatory lesion counts (including nose) ateach evaluation for the per-protocol population. Baseline Week 4 Week 8Week 12 Vehicle Gel (N = 46) Mean 47.7 44.8 46.8 48.2 SD 14.17 23.7732.83 34.97 Median 47.5 40.0 36.5 40.0 Min. to Max. 25 to 70  10 to 1169 to 152 10 to 176 Nitricil ™ NVN1 1% Gel (N = 42) Mean 52.5 44.3 42.139.9 SD 13.49 18.46 29.01 20.72 Median 54.5 41.0 37.0 39.0 Min. to Max.29 to 70 13 to 89 8 to 162 10 to 110 Nitricil ™ NVN1 4% Gel (N = 43)Mean 51.0 40.2 38.2 37.2 SD 14.70 22.57 22.18 28.76 Median 52.0 37.037.0 30.0 Min. to Max. 25 to 70  5 to 112 4 to 90   3 to 131

A mean percentage reduction for inflammatory lesions over time was seenin the PP population for both Nitricil™ NVN1 1% Gel and Nitricil™ NVN14% Gel compared to the Vehicle Gel (FIG. 11). FIG. 11 shows thatseparation from the Vehicle Gel group occurred at week 4 for both theNitricil™ NVN1 1% Gel and Nitricil™ NVN1 4% Gel groups. At week 4, theVehicle Gel group had a 14% mean percentage reduction for inflammatorylesions, whereas the Nitricil™ NVN1 1% Gel group had a 26% meanpercentage reduction for inflammatory lesions and the Nitricil™ NVN1 4%Gel group had a 31% mean percentage reduction for inflammatory lesions.

For the PP population at week 12, the median percent reduction ininflammatory lesions was as follows: 56% for the Nitricil™ NVN1 1% Gelgroup, 66% for the Nitricil™ NVN1 4% Gel group, and 42% for the VehicleGel group. Thus, the median percent reduction in inflammatory lesionsfor the PP population was higher than the mean percent reduction ininflammatory lesions for the PP population for each group. For theNitricil™ NVN1 4% Gel group, 20 of the 41 patients completing the studyhad a reduction in inflammatory lesions of greater than 70%. Theinflammatory lesion count for the PP population is provided in Table 15.

TABLE 15 Summary of inflammatory lesion counts at each evaluation forthe per-protocol population. Baseline Week 4 Week 8 Week 12 Vehicle Gel(N = 46) Mean 28.9 25.6 22.1 19.2 SD 6.11 20.14 21.34 20.04 Median 28.021.5 17.0 17.0 Min. to Max. 20 to 40  4 to 127  3 to 127  0 to 127Nitricil ™ NVN1 1% Gel (N = 42) Mean 29.4 21.4 17.2 14.7 SD 5.91 8.6510.91 10.15 Median 28.0 20.5 17.0 13.5 Min. to Max. 22 to 42 8 to 48 0to 48 1 to 48 Nitricil ™ NVN1 4% Gel (N = 43) Mean 29.3 20.6 16.9 12.7SD 5.00 12.20 11.19 10.10 Median 29.0 18.0 15.0 10.0 Min. to Max. 20 to41 4 to 61 0 to 54 2 to 47

Compared to the Vehicle Gel group, the Nitricil™ NVN1 1% Gel group waseffective in lowering the number of noninflammatory and inflammatorylesions (including and excluding nose), and the Nitricil™ NVN1 4% Gelgroup was effective in lowering the number of noninflammatory lesions(including and excluding nose) and inflammatory lesions. The Nitricil™NVN1 4% Gel group demonstrated statistically significant differencescompared to the Vehicle Gel group, and the Nitricil™ NVN1 4% Gel groupdiffered from the Vehicle group on primary and secondary endpoints. BothNitricil™ NVN1 1% Gel and Nitricil™ NVN1 4% Gel were safe and welltolerated, and no safety signals were identified.

Example 8

Three American Board of Dermatology certified dermatologists withexperience in conducting acne clinical studies conducted a post-hocInvestigator's Global Assessment (IGA) analysis based on the studydescribed in Example 7. The purpose of this analysis was to haveinvestigators with experience in conduct of clinical trials for U.S.approval evaluate the results of treatment. The IGA scoring descriptionfor Examples 7 and 8 is provided in Table 16. The definition of“success” is a score at end of treatment of “0” (clear) or “1” (almostclear) and a minimum 2 grade change from baseline.

TABLE 16 IGA scoring description used for the study described inExamples 7 and 8. Grade Description 0 Clear skin with no inflammatory ornon-inflammatory lesions. 1 Almost clear; rare non-inflammatory lesionswith no more than one inflammatory lesion. 2 Mild severity; greater thanGrade 1; some non-inflammatory lesions with no more than a fewinflammatory lesions (papules/pustules only, no nodular lesions). 3Moderate severity; greater than Grade 2; up to many non-inflammatorylesions and may have some inflammatory lesions, but no more than onesmall nodular lesion 4 Severe; greater than Grade 3; up to manynon-inflammatory and inflammatory lesions, but no more than a fewnodular lesions

The three board-certified dermatologists conducted a blind review of theclinical images available for a subject at both Baseline and Week 12 and“scored” the images taken at Baseline and Week 12. The results areprovided in Table 17 below. All three dermatologists judged theNitricil™ NVN1 Gel, particularly the Nitricil™ NVN1 4% Gel, to haveactivity in the treatment of acne vulgaris.

TABLE 17 Post-hoc analysis results. Vehicle Gel Nitricil ™ NVN1Nitricil ™ NVN1 (N = 44) 1% Gel (N = 44) 4% Gel (N = 41) Dermatologist 1≧2 Grade change 3 5 12 at end of treatment (6.8%) (11.4%) (29.3%)“Success” 2 3 5 (4.5%) (6.8%) (12.2%) Dermatologist 2 ≧2 Grade change 54 10 at end of treatment (11.4%) (9.1%) (24.4%) “Success” 1 0 4 (2.3%)(0.0%) (9.8%) Dermatologist 3 ≧2 Grade change 5 6 13 at end of treatment(11.6%) (13.6%) (31.7%) “Success” 2 3 5 (4.7%) (6.8%) (12.2%)

Example 9

Cosmetically elegant compositions comprising benzoyl peroxide wereprepared. Each composition contained two parts, one part was a benzoylperoxide gel containing a carboxypolymethylene and the second part was acomposition containing a cellulose. Each part was filled into onechamber of a YonWoo 2×15 mL dual chamber pump. The benzoyl peroxide gelformulations are provided in Tables 18 and 19 and the cellulosecomposition for separate combination with each of the two benzoylperoxide gel formulations is provided in Table 20.

TABLE 18 Benzoyl peroxide gel formulation 1. g/batch Ingredient % w/w(Calculated) Water 35.0 175.0 Ethanol, Absolute 30.0 150.0 HexyleneGlycol 12.0 60.0 Benzoyl Peroxide, 75% 6.7 33.5 (Luperox ® A75FP)*Carbopol ® 974P 1.0 5.0 EDTA, Disodium 0.1 0.5 Trolamine QS to pH 6 QSto pH 6 Water QS QS Total 100.00 500.0 *The amount of benzoyl peroxidewas adjusted to yield a final concentration of 5%.

TABLE 19 Benzoyl peroxide gel formulation 2. g/batch Ingredient % w/w(Calculated) Water 33.0 165.0 Ethanol, Absolute 30.0 150.0 Transcutol ®P 12.0 60.0 Benzoyl Peroxide, 75% 6.7 33.5 (Luperox ® A75FP)* Labrasol ®6.0 30.0 Cyclomethicone 5.0 25.0 Klucel ® MF Pharm 1.0 5.0 Carbopol ®974P 0.6 3.0 Phenoxyethanol 1.0 5.0 EDTA, Disodium 0.1 0.5 Trolamine QSto pH 6 QS to pH 6 Water QS QS Total 100.0 500.0 *The amount of benzoylperoxide was adjusted to yield a final concentration of 5%.

TABLE 20 Cellulose composition for separate combination with each of thebenzoyl peroxide formulations. Ingredient % w/w Isopropyl alcohol 85.5Hexylene glycol 10.00 Cyclomethicone 2.50 Hydroxylpropyl cellulose 2.00

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein. Allpublications, patent applications, patents, patent publications, andother references cited herein are incorporated by reference in theirentireties for the teachings relevant to the sentence and/or paragraphin which the reference is presented.

1.-42. (canceled)
 43. A method of treating acne vulgaris, the methodcomprising topically applying to the skin of a subject a compositioncomprising: a first viscosity increasing agent; at least one polyhydricalcohol; at least one buffering agent; a second viscosity increasingagent; at least one organic solvent; at least one humectant; at leastone water repellent; at least one active pharmaceutical ingredient; andwater.
 44. The method of claim 43, wherein the composition is bufferedto a pH of about 3 to about
 8. 45. The method of claim 43, wherein theat least one active pharmaceutical ingredient comprises a nitricoxide-releasing active pharmaceutical ingredient.
 46. The method ofclaim 45, wherein the nitric oxide-releasing active pharmaceuticalingredient comprises a nitric oxide-releasing compound having adiazeniumdiolate functional group, optionally wherein the nitric oxidereleasing compound comprises a NO-releasing co-condensed silicaparticle.
 47. The method of claim 46, wherein the nitric oxide-releasingactive pharmaceutical ingredient is present in the composition in anamount of about 0.5% to about 10% by weight of the composition.
 48. Themethod of claim 46, wherein the composition stores and/or releasesnitric oxide in an amount of about 0.05% to about 3% by weight of thecomposition.
 49. The method of claim 48, wherein the one nitricoxide-releasing active pharmaceutical ingredient releases nitric oxidein an amount of at least about 50% over a period of time of about 3hours or less after topical application of the composition to skin of asubject.
 50. The method of claim 43, wherein the composition comprises afirst composition in admixture with a second composition, the firstcomposition comprising: the at least one polyhydric alcohol in an amountof about 1% to about 30% by weight of the first composition; the atleast one viscosity increasing agent in an amount of about 0.1% to about5% by weight of the first composition; the at least one buffering agentin an amount of about 0.01% to about 2% by weight of the firstcomposition; and water in an amount of about 70% to about 99% by weightof the first composition; and the second composition comprising: thesecond viscosity increasing agent in an amount of about 0.5% to about30% by weight of the second composition; at least one organic solvent inan amount of about 50% to about 90 by weight of the second composition;at least one humectant in an amount of about 2% to about 20% by weightof the second composition; and the at least one water repellent in anamount of about 0.5% to about 15% by weight of the second composition.51. The method of claim 43, wherein the composition is cosmeticallyelegant.
 52. The method of claim 43, wherein the method reduces P. acnescounts on the skin of the subject.
 53. The method of claim 52, whereinP. acnes counts are reduced by about 10% or greater over a definedperiod of time.
 54. The method of claim 52, wherein the reduction in P.acnes counts is within 12 weeks or less.
 55. A method of reducinginflammatory and/or noninflammatory lesions present on the skin of asubject, the method comprising topically applying to the skin of thesubject a composition comprising: a first viscosity increasing agent; atleast one polyhydric alcohol; at least one buffering agent; a secondviscosity increasing agent; at least one organic solvent; at least onehumectant; at least one water repellent; at least one activepharmaceutical ingredient; and water.
 56. The method of claim 55,wherein the composition is buffered to a pH of about 3 to about
 8. 57.The method of claim 55, wherein the active pharmaceutical ingredientcomprises a nitric oxide-releasing active pharmaceutical ingredient. 58.The method of claim 57, wherein the nitric oxide-releasing activepharmaceutical ingredient comprises a nitric oxide-releasing compoundhaving a diazeniumdiolate functional group, optionally wherein thenitric oxide releasing compound comprises a NO-releasing co-condensedsilica particle.
 59. The method of claim 58, wherein the nitricoxide-releasing active pharmaceutical ingredient is present in thecomposition in an amount of about 0.5% to about 10% by weight of thecomposition.
 60. The method of claim 58, wherein the composition storesand/or releases nitric oxide in an amount of about 0.05% to about 3% byweight of the composition.
 61. The method of claim 60, wherein the onenitric oxide-releasing active pharmaceutical ingredient releases nitricoxide in an amount of at least about 50% over a period of time of about3 hours or less after topical application of the composition to skin ofa subject.
 62. The method of claim 55, wherein the composition comprisesa first composition in admixture with a second composition, the firstcomposition comprising: the at least one polyhydric alcohol in an amountof about 1% to about 30% by weight of the first composition; the atleast one viscosity increasing agent in an amount of about 0.1% to about5% by weight of the first composition; the at least one buffering agentin an amount of about 0.01% to about 2% by weight of the firstcomposition; and water in an amount of about 70% to about 99% by weightof the first composition; and the second composition comprising: thesecond viscosity increasing agent in an amount of about 0.5% to about30% by weight of the second composition; at least one organic solvent inan amount of about 50% to about 90 by weight of the second composition;at least one humectant in an amount of about 2% to about 20% by weightof the second composition; and the at least one water repellent in anamount of about 0.5% to about 15% by weight of the second composition.63. The method of claim 55, wherein the composition is cosmeticallyelegant.
 64. The method of claim 55, wherein the number of inflammatoryand/or noninflammatory lesions is reduced by about 10% or greater over adefined period of time.
 65. The method of claim 55, wherein thereduction in inflammatory and/or noninflammatory lesions is within 12weeks or less.
 66. A composition in the form of a hydrogel, the hydrogelhaving a pH of about 3 to about 8 and is compatible with a secondcomposition to form a structure in which an active pharmaceuticalingredient is suspended, upon combination of the hydrogel and the secondcomposition, the hydrogel comprising: at least one polyhydric alcohol;at least one viscosity increasing agent; at least one buffering agent;and water.
 67. The composition of claim 66, wherein the hydrogelcomprises: the at least one polyhydric alcohol in an amount of about 1%to about 30% by weight of the hydrogel; the at least one viscosityincreasing agent in an amount of about 0.1% to about 5% by weight of thehydrogel; the at least one buffering agent in an amount of about 0.01%to about 2% by weight of the hydrogel; and water in an amount of about70% to about 99% by weight of the hydrogel;
 68. The composition of claim66, wherein, upon combination, the hydrogel and the second compositionprovide a combined composition having a pH in a range of about 6 toabout
 8. 69. The composition of claim 66, wherein, upon combination, thehydrogel and the second composition provide a combined composition thatis cosmetically elegant.
 70. The composition of claim 66, wherein theactive pharmaceutical ingredient is a nitric oxide releasing activepharmaceutical ingredient and the hydrogel modulates the release ofnitric oxide from the NO releasing active pharmaceutical ingredient. 71.A method of treating acne vulgaris, the method comprising: providing ananhydrous topical gel comprising a nitric oxide-releasing activepharmaceutical ingredient; providing a hydrogel; combining the anhydroustopical gel comprising the nitric oxide-releasing active pharmaceuticalingredient and the hydrogel to provide a nitric oxide-releasing topicalcomposition; and topically applying to the skin of a subject the nitricoxide-releasing topical composition.
 72. The method of claim 71, whereinthe nitric oxide-releasing topical composition is buffered to a pH ofabout 3 to about
 8. 73. The method of claim 71, wherein the nitricoxide-releasing active pharmaceutical ingredient comprises a nitricoxide-releasing compound having a diazeniumdiolate functional group,optionally wherein the nitric oxide releasing compound comprises aNO-releasing co-condensed silica particle.
 74. The method of claim 71,wherein the nitric oxide-releasing active pharmaceutical ingredient ispresent in the composition in an amount of about 0.5% to about 10% byweight of the composition.
 75. The method of claim 71, wherein thenitric oxide-releasing topical composition stores and/or releases nitricoxide in an amount of about 0.05% to about 3% by weight of thecomposition.
 76. The method of claim 71, wherein the nitricoxide-releasing active pharmaceutical ingredient releases nitric oxidein an amount of at least about 50% over a period of time of about 3hours or less after topical application of the nitric oxide-releasingtopical composition to skin of a subject.